This work was supported by a Joseph P. Healey grant from the University of Massachusetts Boston (L.S.H.) and R15 GM86805 from the NIH (L.S.H.). S.M.P. is supported in part by a Sanofi-Genzyme Fellowship at the University of Massachusetts Boston.

1. Preparing for Sporulation
Note: The media described in this protocol are made using standard recipes and methods13,17. Table 1 gives the formulation for 1 L of the various media used in this protocol.
<table border="1" cellpadding="0" cellspacing="0" height="180" width="469">
	<colgroup>
		<col />
		<col />
		<col span="3" />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="15">
			<td height="15" style="height:20px;"></td>
			<td style="width: 55px;">Bacto Peptone</td>
			<td style="width: 50px;">Yeast Extract</td>
			<td style="width: 50px;">Bacto Agar</td>
			<td style="width: 60px;">Dextrose</td>
			<td style="width: 70px;">Potassium Acetate</td>
			<td style="width: 60px;">Glycerol</td>
			<td style="width: 60px;">ddH2O</td>
		</tr>
		<tr height="15">
			<td height="15" style="height:20px;">YPG plates</td>
			<td>20 g</td>
			<td>10 g</td>
			<td>20 g</td>
			<td>-</td>
			<td>-</td>
			<td>30 ml</td>
			<td>970 ml</td>
		</tr>
		<tr height="15">
			<td height="15" style="height:20px;">YPD plates</td>
			<td>20 g</td>
			<td>10 g</td>
			<td>20 g</td>
			<td>20 g</td>
			<td>-</td>
			<td>-</td>
			<td>1,000 ml</td>
		</tr>
		<tr height="15">
			<td height="15" style="height:20px;">YPD liquid</td>
			<td>20 g</td>
			<td>10 g</td>
			<td>-</td>
			<td>20 g</td>
			<td>-</td>
			<td>-</td>
			<td>1,000 ml</td>
		</tr>
		<tr height="15">
			<td height="15" style="height:20px;">YPA liquid</td>
			<td>20 g</td>
			<td>10 g</td>
			<td>-</td>
			<td>-</td>
			<td>20 g</td>
			<td>-</td>
			<td>1,000 ml</td>
		</tr>
		<tr height="15">
			<td style="height: 20px; width: 80px;">Sporulation media</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>10 g</td>
			<td>-</td>
			<td>1,000 ml</td>
		</tr>
	</tbody>
</table>
Table 1: Media formulations. Amounts are given for 1 L of media. Specifics about media ingredients can be found in the Materials Table.
<ol>
	<li>If All Sporulating Cells will be the Same Genotype, Follow This Procedure:
	<ol>
		<li>Thaw yeast strain to be used onto a YPG (YPGlycerol) plate. Grow yeast for 12-16 hr at 30 &#176;C. Importantly, SK1 cells are also poised to sporulate, so SK1 strains should not be left too long on YPG plates, or they may sporulate on the plate. 
		Note: A freshly thawed strain grown on YPG is preferred, as utilization of glycerol as a carbon source requires oxidative phosphorylation, and thus selects for cells that have functional mitochondrial DNA. SK1 cells have a tendency to lose functional mitochondrial DNA and become petites. Functional mitochondrial DNA is needed for sporulation.</li>
		<li>Streak yeast from the YPG plate onto a YPD (YPDextrose) plate for single colonies. Grow yeast on YPD plate 24-28 hr at 30 &#176;C.</li>
		<li>After about 36-48 hr of growth on YPD solid media, start a 25 ml YPD culture using a single colony from the YPD plate. Grow this culture until cells are about OD600 = 4.0 to 7.0. Perform this step at 30 &#176;C overnight in a shaking incubator, shaking at 220 rpm.</li>
		<li>From the YPD culture, use an appropriate amount of cells to start 1,000 ml of YPA (YPAcetate) culture that is OD600 = 0.1 for each 96 well plate need in Section 2. Grow yeast in a 30 &#176;C shaking incubator (shaking at 220 rpm) until the culture OD600 is between 1.3 to 1.5. 
		Note: Inoculate 100 ml of YPA culture for each 96 multiwell plate to be screened in Section 2. For example, if screening four 96 multiwell plates, 400 ml of YPA culture will be needed. Starting with 10% extra volume in the YPA culture is necessary to protect against evaporative volume loss. Typically, it will take between 12-16 hr for cells to reach the appropriate OD600; the amount of time may vary depending on the genotype of the yeast strain. YPA is the presporulation media. Pregrowth in acetate helps induce expression of genes (such as IME1) needed for efficient and synchronous sporulation6.</li>
		<li>Spin at 1,800 x g in a table top centrifuge for 5 min to pellet cells grown in YPA. Wash cells 1x with 0.5 volume of sterile autoclaved deionized water. Spin again, and discard wash. Resuspend cells in 1x volume sporulation media. Continue to Section 2.</li>
	</ol>
	</li>
	<li>If using cells for sporulation that are of different genotypes, (i.e., transformed with different plasmids or containing different mutations, typically obtained and stored as frozen stocks), array cells of the different genotypes into a 96 multiwell format. Then, follow this procedure:
	<ol>
		<li>Use a sterile 96 prong frogger to transfer cells from thawed frozen stocks in 96 multiwell plate(s) onto YPG solid media. Grow yeast overnight at 30 &#176;C.</li>
		<li>Using a sterile 96 prong frogger, transfer cells from YPG solid media onto YPD solid media or selective solid media (i.e., synthetic minimal media lacking amino acids, if the cells contain plasmids that require selection). Grow yeast at 30 &#176;C until colonies are formed for each strain, typically 1 day on YPD and 2 days if selective media is necessary.</li>
		<li>Aliquot 1.2 ml of YPD liquid media or selective liquid media into each well of a 96 deep 2 ml multiwell plate. Transfer cells from solid media into liquid media using a frogger, cover with a plastic lid held in place by tape, and grow overnight in 30 &#176;C shaker, shaking at 220 rpm. 
		Note: To minimize contamination, cover plates using the plastic lids from a rectangular petri plate. Hold lids in place using a piece of tape, so that air circulation is minimally restricted.</li>
		<li>Centrifuge plates at 1,800 x g for 5 min, using a swinging bucket centrifuge with adaptors to accommodate 96 well plates. Pour off media from cells; add 500 &#181;l YPA to each well and resuspend pelleted cells in the YPA. Cover with plastic lid held in place by tape.</li>
		<li>Grow cells for 15 hr at 30 &#176;C in a shaking incubator, shaking at 220 rpm. Continue to Section 2.</li>
	</ol>
	</li>
</ol>
2. Sporulating Cells in a 96 Multiwell Format
<ol>
	<li>Add either one 3 mm sterile solid glass bead or a sterile 5 mm x 2 mm magnetic stir bar into each well of a 96 well plate with 1.3 ml wells. (See Representative Results below for a discussion on whether to use a bead or a stir bar.)</li>
	<li>If all cells to be screened are of the same genotype, go to 2.2.1. If cells to be screened are of different genotypes, go to 2.2.2.
	<ol>
		<li>Take cells from step 1.1.5 and aliquot 500 &#181;l of cells plus media into each well of the 96 multiwell plate. Cover with plastic lid held in place by tape. Proceed to 2.3.</li>
		<li>Spin cells that are in YPA from 1.2.4 at 1,800 x g for 5 min, using a swinging bucket centrifuge with adaptors to accommodate 96 well plates. Pour off media from cells. Add 500 &#181;l of sporulation media to each well and resuspend pelleted cells in the sporulation media. Cover with plastic lid held in place by tape. Proceed to 2.3.</li>
	</ol>
	</li>
	<li>If using a glass bead, place samples in a shaking incubator at 30&#176; C and shake at 220 rpm for sporulation. If using a magnetic stir bar, place samples on a stir plate inside of a 30 &#176;C incubator for sporulation. 
	Note: The exact stir speed for the magnetic stir bars is not very important, as long as all bars are moving energetically and the culture is aerated.</li>
	<li>To monitor progression through sporulation, withdraw samples from the sporulating culture for visualization, Add 6 &#181;l of cells from the sporulating cultures to 24 &#181;l of sporulation media in a 96 well coverslip plate (1:5 dilution). Leave cells to settle for 5 min before examining cells using an inverted microscope. 
	Note: In wild type SK1 cells, compact mature spores will form by 36 hours after transfer to sporulation media. Progression through sporulation can be monitored by microscopy using fluorescent markers, such as a nuclear marker (i.e., Htb2-mCherry18) to examine the meiotic division or a prospore membrane marker (i.e., G2019) to examine spore morphogenesis. Alternatively, the formation of refractile spores can be seen using Nomarski DIC microscopy starting about 12 hr after transfer to sporulation media.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+signaling+pathways+orchestrate+yeast+development.">Pleiotropic signaling pathways orchestrate yeast development.</a> Curr. Opin. Microbiol. 14 (6), 676-681 (2011).</li><li>Broach, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+control+of+growth+and+development+in+yeast.">Nutritional control of growth and development in yeast.</a> Genetics. 192 (1), 73-105 (2012).</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> Microbiol. Mol. 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The authors have no competing financial interests.

Here we present a protocol for sporulating SK1 yeast in a 96 multiwell format. Aeration is key for efficient sporulation, which requires the use of either a stir bar or a glass bead in each well. When cells are sporulated in a 96 multiwell plate in a shaking incubator without either a bead or a stir bar, cells do not sporulate efficiently. Only a small increase in sporulation efficiency is seen when cells are sporulated without either a bead or a stir bar in a shaking incubator, compared to being at 30 &#176;C without agitation (Table 1; 17% in shaker vs 10% placed on a stir plate). Similarly, the use of a 7 mm stir bar resulted in poor sporulation efficiency (28%), likely because the 7 mm stir bars did not aerate the culture properly due to interactions with stir bars in adjacent wells. This protocol describes conditions for sporulation using the synchronously and efficiently sporulating SK1 yeast strain7,8 used commonly to study sporulation; sporulation efficiencies that can be obtained with other yeast strains using this protocol should be examined before undertaking a large screen using these techniques.
Although a 5 mm x 2 mm stir bar results in a slightly better sporulation compared to the use of a glass bead (66% with 5 mm x 2 mm stir bar vs 56% with a glass bead), the cost required to purchase enough stir bars for each well can be prohibitive. The sporulation efficiencies obtained using a glass bead provides a low-cost solution that creates enough aeration for high-throughput screening in a 96 multiwell format16. Unfortunately, the addition of a stir bar or a glass bead did not achieve the very high sporulation efficiencies (typically ~80% or greater) seen when sporulating in flasks, and thus subtle sporulation phenotypes may be difficult to detect when screening in a 96 well format.
Unlike previously described sporulation methods9-13 which utilize solid media or larger liquid media volumes using flasks or culture tubes, this protocol describes conditions for efficient sporulation in a 96 multiwell format. The use of a 96 multiwell format is compatible with different types of high throughput screens that can be carried out to examine sporulation phenotypes. For example, chemical screens that affect processes in sporulation can be carried out using this technique, where sporulating cells are treated with different compounds that have been arrayed into a 96 well format. In this case, the cells to be sporulated would all be of the same genotype, and the compounds would be added at step 2.2 of sporulation. This protocol is also compatible with screening cells of different genotypes for sporulation phenotypes, such as yeast cells transformed with a high copy library or a yeast mutant strain collection. For this case, the strains would need to be arrayed first in a 96 well format and the protocol described starting at step 1.2 would be appropriate.

Sporulation in the budding yeast has been studied to provide insights into many aspects of biology, including the control of chromosome segregation during meiosis1, mechanisms of genetic recombination2, the control of development by cell signaling3, nutritional control of development4, the transcriptional regulation of development5, and the examination of spore formation6. Spore formation includes a novel cell division event involving the formation of new membrane compartments within the mother cell followed by the deposition of a protective spore wall6. These studies that examine sporulating cells often take advantage of the rapidly sporulating yeast strain SK1, which can undergo the process of sporulation in about 24 hr in a relatively efficient fashion7,8. Although optimization of sporulation conditions for budding yeast have been described9-13, these experiments examined sporulation on solid media or in larger scale liquid cultures where sporulation is carried out using culture tubes or flasks.
Here we describe a method for sporulating yeast in a 96 multiwell plate format. We find that for this method, aeration is critical for synchronous and efficient sporulation, and have devised techniques to ensure sufficient sporulation a small-volume multiwell format. Sporulating in a 96 multiwell plate format allows for cells to be screened using high-throughput techniques and reagents optimized for a multiwell plate format, such screening for high copy suppressors using a tiled library14-16.

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nunc 1.3 ml DeepWell Plates</td>
			<td style="width:123px;">ThermoScientific</td>
			<td style="width:119px;">260251</td>
			<td style="width:288px;">Used for sporulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nunc 2.0 ml DeepWell plates</td>
			<td style="width:123px;">ThermoScientific</td>
			<td>278743</td>
			<td>Used for presporulation growth, step 1.2.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 mm glass bead</td>
			<td style="width:123px;">Fisher</td>
			<td>11-312A</td>
			<td>Used for sporulation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5 mm x 2 mm stir bar, pack of 12</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">14-511-82</td>
			<td>Used for sporulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96 well frogger</td>
			<td style="width:123px;">V&#38;P Scientific</td>
			<td style="width:119px;">VP407</td>
			<td>needed for step 1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">library copier</td>
			<td style="width:123px;">V&#38;P Scientific</td>
			<td style="width:119px;">VP381</td>
			<td>needed for step 1.2; to be used with the frogger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rectangular petri dish</td>
			<td style="width:123px;">ThermoScientific</td>
			<td style="width:119px;">264728</td>
			<td>needed for step 1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Peptone</td>
			<td style="width:123px;">BD</td>
			<td>211677</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
			<td style="width:123px;">BD</td>
			<td>212750</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
			<td style="width:123px;">BD</td>
			<td style="width:119px;">212750</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dextrose</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">D16-3</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Acetate</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">P171-500</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">G33-500</td>
			<td>needed for media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Black 96 well glass bottom plate</td>
			<td style="width:123px;">MatTek</td>
			<td style="width:119px;">PBK96G-1.5.5-F</td>
			<td>needed for step 2.4</td>
		</tr>
	</tbody>
</table>

efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

To assess this protocol, sporulation efficiencies obtained from sporulating cells in multiwell plates (as described above) were compared to cells sporulated using larger volumes in flasks (Table 2). The use of multiwell plates did not achieve the high efficiency seen when sporulating in flasks, where ~80% efficiency can be routinely seen. Sporulating in multiwell plates with proper aeration (provided by glass beads or stir bars) can achieve sufficient sporulation efficiencies greater than 50%, with the best results (66% efficiency) obtained using a 5 mm x 2 mm stir bar. A representative sporulation of two different strains (wild type and smk1&#916;) is shown here (Figure 1). These cells are examined after 36 hr in sporulation media and visualized using a 96 well glass bottom plate.
<img alt="Figure 1" src="/files/ftp_upload/54584/54584fig1.jpg" /> 
Figure 1: Sporulating cells from a representative sporulation. Wild type and smk1&#916; cells20 sporulated in a 96 multiwell plates. Cells were visualized using a 63X objective on an inverted microscope. Refractile tetrads (arrowheads) can be seen in the wild type culture, but not in the culture containing smk1&#916; cells. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54584/54584fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Sporulation was tested using either a 5 mm or 7 mm stir bar, each of which fits within the well of a 96 1.3 ml multiwell plate (Table 2). Using a 7 mm stir bar resulted in poor sporulation efficiency (28%) compared to the higher efficiency (66%) achieved using the 5 mm stir bar. The 7 mm stir bar in a well tended to interact with a stir bar in an adjacent well (Figure 1), preventing the bars from being able to properly stir and provide adequate aeration of the culture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>sporulating cells (%)</td>
			<td>SEM&#160; (%)</td>
		</tr>
		<tr>
			<td>flask in shaker</td>
			<td>82</td>
			<td>1.8</td>
		</tr>
		<tr>
			<td>no bead in shaker</td>
			<td>17</td>
			<td>2.4</td>
		</tr>
		<tr>
			<td>bead in shaker</td>
			<td>56</td>
			<td>3</td>
		</tr>
		<tr>
			<td>no stir bar on stir plate</td>
			<td>10</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>5 mm stir bar on stir plate</td>
			<td>66</td>
			<td>3.9</td>
		</tr>
		<tr>
			<td>7 mm stir bar on stir plate</td>
			<td>28</td>
			<td>6.7</td>
		</tr>
		<tr>
			<td>SEM = standard error of the mean</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Sporulation efficiencies using different conditions. Sporulation was conducted in 96 well multiwell plates as described above, or, if noted, in 500 ml flasks containing 50 ml of sporulation media. Eight different wells were averaged for each condition carried out in a multiwell plate. Three different flasks were sporulated. All cultures are technical replicates, started from the same colony of mCherry-tagged Htb2 but otherwise wild type diploid SK1 yeast strain (LH90218). These cells were grown in a single flask of YPD and a single flask of YPA, and then divided to inoculate 96-well plates containing sporulation media, as described above. Sporulation efficiency was assayed by counting refractile spores 36 hr after transfer to sporulation media using Nomarski DIC brightfield microscopy.
<img alt="Figure 2" src="/files/ftp_upload/54584/54584fig2.jpg" /> 
Figure 2: Stir bars in 96 1.3 ml multiwell plates. Sporulation culture containing either (A) 5 mm x 2 mm stir bars (red) or (B) 7 mm x 2 mm stir bars (blue) in a 96 1.3 ml multiwell plate on a magnetic stir plate. Scale bar = 10 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54584/54584fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

efficient-sporulation-saccharomyces-cerevisiae-96-multiwell

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Biology

10.0K Views.  University of Massachusetts Boston. Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Video: Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.