Name: microscopy of fission yeast sexual lifecycle
Uploaded: 2016-03-09T15:00:00
Description: Watch this Scientific Journal Video about Microscopy of Fission Yeast Sexual Lifecycle at JoVE.com

AV was supported by an EMBO long-term postdoctoral fellowship. Research in the Martin lab is funded by an ERC Starting grant (GeometryCellCycle) and a Swiss National Science Foundation grant (31003A_155944) to SGM.

Microscopy analysis of fission yeast sexual reproduction
1. Media Preparation
<ol>
	<li>Prepare Minimum Sporulation medium (MSL-N)15 by mixing the following components: Glucose: 10 g/L, KH2PO4: 1 g/L, NaCl: 0.1 g/L, MgSO4&#183;7H2O: 0.2 g/L. Add Trace elements (10,000x): 100 &#181;l/L, Vitamins (1,000x): 1 ml/L, and 0.1 M CaCl2&#160;ml/L. Filter-sterilize using a 0.22 &#956;m pore size filter and store at room temperature (RT).
	<ol>
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<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+learned+from+studies+of+fission+yeast+mating-type+switching+and+silencing.">Lessons learned from studies of fission yeast mating-type switching and silencing.</a> Annu Rev Genet. 41, 213-236 (2007).</li><li>Beach, D. H., Klar, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearrangements+of+the+transposable+mating-type+cassettes+of+fission+yeast.">Rearrangements of the transposable mating-type cassettes of fission yeast.</a> EMBO J. 3 (3), 603-610 (1984).</li><li>Merlini, L., Dudin, O., Martin, S. 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O., Martin, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42+explores+the+cell+periphery+for+mate+selection+in+fission+yeast.">Cdc42 explores the cell periphery for mate selection in fission yeast.</a> Curr Biol. 23 (1), 42-47 (2013).</li><li>Dudin, O., 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=A+formin-nucleated+actin+aster+concentrates+cell+wall+hydrolases+for+cell+fusion+in+fission+yeast.">A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast.</a> J Cell Biol. 208 (7), 897-911 (2015).</li><li>Chikashige, Y., 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=Telomere-led+premeiotic+chromosome+movement+in+fission+yeast.">Telomere-led premeiotic chromosome movement in fission yeast.</a> Science. 264 (5156), 270-273 (1994).</li><li>Bahler, J., 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=Heterologous+modules+for+efficient+and+versatile+PCR-based+gene+targeting+in+Schizosaccharomyces+pombe.">Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.</a> Yeast. 14 (10), 943-951 (1998).</li><li>Egel, R., Willer, M., Kjaerulff, S., Davey, J., Nielsen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+pheromone+production+and+response+in+fission+yeast+by+a+halo+test+of+induced+sporulation.">Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation.</a> Yeast. 10 (10), 1347-1354 (1994).</li><li>Tran, P. T., Marsh, L., Doye, V., Inoue, S., Chang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+for+nuclear+positioning+in+fission+yeast+based+on+microtubule+pushing.">A mechanism for nuclear positioning in fission yeast based on microtubule pushing.</a> J Cell Biol. 153 (2), 397-411 (2001).</li><li>Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., Waldo, 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=Engineering+and+characterization+of+a+superfolder+green+fluorescent+protein.">Engineering and characterization of a superfolder green fluorescent protein.</a> Nat Biotechnol. 24 (1), 79-88 (2006).</li><li>Kitamura, K., Shimoda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Schizosaccharomyces+pombe+mam2+gene+encodes+a+putative+pheromone+receptor+which+has+a+significant+homology+with+the+Saccharomyces+cerevisiae+Ste2+protein.">The Schizosaccharomyces pombe mam2 gene encodes a putative pheromone receptor which has a significant homology with the Saccharomyces cerevisiae Ste2 protein.</a> EMBO J. 10 (12), 3743-3751 (1991).</li><li>Tanaka, K., Davey, J., Imai, Y., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schizosaccharomyces+pombe+map3++encodes+the+putative+M-factor+receptor.">Schizosaccharomyces pombe map3+ encodes the putative M-factor receptor.</a> Mol Cell Biol. 13 (1), 80-88 (1993).</li><li>Motegi, F., Arai, R., Mabuchi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+type+V+myosins+in+fission+yeast,+one+of+which+functions+in+polarized+cell+growth+and+moves+rapidly+in+the+cell.">Identification of two type V myosins in fission yeast, one of which functions in polarized cell growth and moves rapidly in the cell.</a> Mol Biol Cell. 12 (5), 1367-1380 (2001).</li><li>Sajiki, K., Pluskal, T., Shimanuki, M., Yanagida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+analysis+of+fission+yeast+at+the+onset+of+nitrogen+starvation.">Metabolomic analysis of fission yeast at the onset of nitrogen starvation.</a> Metabolites. 3 (4), 1118-1129 (2013).</li><li>Miyawaki, A., Nagai, T., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+protein+fluorophore+formation+and+engineering.">Mechanisms of protein fluorophore formation and engineering.</a> Curr Opin Chem Biol. 7 (5), 557-562 (2003).</li><li>Dyer, J. 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Environmental conditions, and nutrient availability in particular, strongly affect the fission yeast physiology. Nitrogen starvation is necessary for commitment to the sexual reproduction and initially leads to striking changes in the mitotic cell cycle progression (Ref.21 and Figure 2). Upon nitrogen removal from exponentially growing population, cell size at division rapidly decreases (Figure 2C) and the majority of cells arrest mitotic progression shorter than the length of...

Though genetic exchange between two cells is the central event in sexual reproduction, it relies on a chain of events that promote cell differentiation, allow for partner choice, carry out cell-cell fusion and maintain genomic stability. Thus the sexual lifecycle presents itself as a model system to study a number of biological questions regarding developmental switches, response to extrinsic stimuli, plasma membrane fusion, chromosome segregation, etc. Exploring the fission yeast sexual cycle to study these phenomena brings the benefits of the model system&#39;s powerful genetics, well-established high-throughput approaches and sophisticated microscopy. Sex in fission yeast is a heterotypic event between a P-cell and an M-cell of distinct mating types. The two cell types differentially express a number of genes1,2 including those for production of the secreted P- and M-pheromones, pheromone-receptors Map3 and Mam2 as well as pheromone-proteases Sxa1 and Sxa2. Homothallic strains, such as the commonly used h90 strain, carry the genetic information for both mating types in a single genome and cells undergo a complex pattern of mating type switching throughout the mitotic lifecycle (reviewed in Ref.3). Multiple isolates of heterothallic fission yeast that rarely or never switch mating type are also commonly used4, most prominently the h+N (P-type) and h-S (M-type) strains.
In fission yeast, entry into the sexual lifecycle is under strict nutritional regulation. Only nitrogen-starved fission yeast cells arrest mitotic reproduction and produce diffusible pheromones to signal presence of a mating partner and promote further steps of the sexual cycle (reviewed in Ref.5). Nitrogen deprivation de-represses the key transcriptional regulator of mating Ste11 that acts as a developmental switch and promotes expression of mating specific genes including the pheromone receptor and the pheromone production genes6,7. Pheromone-receptor engagement activates the receptor-coupled protein G-alpha and downstream MAPK signaling which further enhances Ste11 transcriptional activity8-10, thus increasing pheromone production in a positive feedback between mating partners. Pheromone levels are crucial to induce different cell polarization states by regulating the master organizer of cell polarity, the Rho-family GTPase Cdc4211. Upon exposure to low pheromone concentrations, active Cdc42 is visualized in dynamic patches exploring the cell periphery, and no cell growth is observed at this stage. Increased pheromone levels promote the stabilization of Cdc42 activity to a single zone and growth of a polarized projection, termed the shmoo, which brings partner cells in contact. Subsequently, the two haploid mating partners fuse to form a diploid zygote. Recent work reveals the existence of a novel actin structure essential for fusion that is assembled by the mating-induced formin Fus112. This fusion focus concentrates type-V myosin dependent processes and positions the cell wall degradation machinery, thus allowing remodeling of the cell wall to permit plasma membrane contact without cell lysis12. Upon cell-cell fusion, the nuclei come in contact and undergo karyogamy. A prominent dynein-dependent back-and-forth movement of the nucleus inside the zygote (the horse-tail movement) then promotes the pairing of chromosome homologs13,14, which is followed by meiosis. Finally, the four products of meiosis are packaged into individual spores during sporulation.
Because of its complexity and the numerous steps involved, detailed monitoring of mating has been challenging. Two notable difficulties are that the entire process takes well over fifteen hours and that cells are difficult to synchronize. These difficulties are circumvented by single-cell microscopy approaches. Here a general protocol to investigate the sexual lifecycle in fission yeast is presented. With minor adjustments, this protocol permits the study of all the different steps of the process, namely the induction of mating gene product, cell polarization and pairing between sister-cells after mating type switching and between non-sister partners, cell-cell fusion, and post-fusion horse-tail movement, meiosis and sporulation. This method allows to 1) easily visualize fluorescently tagged proteins over time pre-, during and post-fusion; 2) discriminate the behavior of cells of opposite mating type; and 3) measure and quantify parameters such as shmooing, mating, fusion or sporulation efficiency.

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			<td height="17" style="height:17px;width:219px;">Glucose</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:108px;">G8270-10KG</td>
			<td style="width:224px;"></td>
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			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>1.05108.0050</td>
			<td></td>
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			<td height="17" style="height:17px;">NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71381</td>
			<td></td>
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			<td height="21" style="height:21px;">MgSO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>63140</td>
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			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>12095</td>
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			<td height="17" style="height:17px;">Pantothenate</td>
			<td>AppliChem</td>
			<td>A2088,0025</td>
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			<td height="17" style="height:17px;">Nicotinic Acid</td>
			<td>AppliChem</td>
			<td>A0963,0100</td>
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			<td height="17" style="height:17px;">Inositol</td>
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			<td>A1716,0100</td>
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			<td>AppliChem</td>
			<td>A0967,0250</td>
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			<td>Sigma-Aldrich</td>
			<td>B6768-1KG</td>
			<td></td>
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			<td height="21" style="height:21px;">MnSO4</td>
			<td>AppliChem</td>
			<td>A1038,0250</td>
			<td></td>
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			<td>Sigma-Aldrich</td>
			<td>Z4750</td>
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			<td height="21" style="height:21px;">FeCl2.6H2O</td>
			<td>AppliChem</td>
			<td>A3514,0250</td>
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			<td height="21" style="height:21px;">Molybdenum oxide (VI) (MoO3)</td>
			<td>Sigma-Aldrich</td>
			<td>69850</td>
			<td></td>
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			<td height="17" style="height:17px;">KI</td>
			<td>AppliChem</td>
			<td>A3872,0100</td>
			<td></td>
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			<td height="21" style="height:21px;">CuSO4.5H2O</td>
			<td>AppliChem</td>
			<td>A1034,0500</td>
			<td></td>
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			<td height="17" style="height:17px;">Citric Acid&#160;</td>
			<td>AppliChem</td>
			<td>A2344,0500</td>
			<td></td>
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			<td height="17" style="height:17px;">Agarose</td>
			<td>Promega</td>
			<td>V3125</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">(NH4)2SO4&#160;</td>
			<td>Merck</td>
			<td>1.01217.1000</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">L-Leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L8000-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Adenine Hemisulfat Salt, mini 99%</td>
			<td>Sigma-Aldrich</td>
			<td>A9126-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Uracil&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>U0750</td>
			<td></td>
		</tr>
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			<td height="17" style="height:17px;">Lanolin</td>
			<td>Sigma-Aldrich</td>
			<td>L7387</td>
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		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vaseline</td>
			<td>Reactolab</td>
			<td>92045-74-4</td>
			<td></td>
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			<td height="17" style="height:17px;">Paraffin</td>
			<td>Reactolab</td>
			<td>7005600</td>
			<td></td>
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microscopy of fission yeast sexual lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

Fission Yeast Growth and Mating Dynamics Upon Removal of a Nitrogen Source 
As nitrogen starvation is a prerequisite for initiation of sexual reproduction in fission yeast, wild-type homothallic h90 strain was monitored upon shift from nitrogen-rich to nitrogen-deprived medium (Figure 2), following the protocol outlined in Figure 1. Briefly, cells were grown O/N to exponential phase (O.D.600 = 0.5) in MSL+N medium, collected, wash...

Watch this Scientific Journal Video about Microscopy of Fission Yeast Sexual Lifecycle at JoVE.com

Microscopy of Fission Yeast Sexual Lifecycle

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

The overall goal of this method is to monitor the entire sexual reproduction process in fission yeast by light microscopy. This method describes the preparation of fission yeast cells for time-lapse imaging of various stages of the sexual lifecycle, such as mating partner choice, polarized growth, cell-cell fusion, myosis, and sporulation. The main advantage of this technique is that it provides single cell visualization and so overcomes the problem that cells do not enter sexual differentiation synchronously.This method is very simple, but the visual demonstration is useful to show how to mount several strains in parallel for microscopy. Demonstrating the procedure with my PhD student Omaya Dudin, will be Laura Merlini, a postdoc in my lab. There are several variations to this protocol which can be tailored to study various stages of the sexual lifecycle, using either homothallic or heterothallic strains.In this demonstration, homothallic H90 strains will be used. In these strains, mating type switching takes place during the last mitotic division after nitrogen deprivation, resulting in a mix of the two mating types. In the evening, two days prior to the mating experiment, inoculate freshly streaked strains from solid media into culture tubes containing 3 milliliters of minimum sporulation medium with nitrogen or MSL plus N.Incubate overnight with shaking at 25 degrees Celsius.On the following morning, if necessary, dilute the cell suspensions in MSL plus N to ensure exponential growth throughout the day. In the evening, measure the optical density and dilute the cells in 20 milliliters of MSL plus N media in 100 milliliter flasks to an optical density of 025 Wild-type cell cultures should reach an optical density 600 of about 8 the following morning. So the dilution should be adjusted accordingly, if working with strains with longer generation times.Incubate the cultures overnight at 30 degrees Celsius with shaking. On the following morning, measure the optical density to verify that each culture has an optical density 600 around 8. Pellet the cells at 1000 x g for one minute and transfer it to a 1.5 milliliter tube.Wash the cells three times in 1 milliliter of minimum sporulation medium without nitrogen or MSL minus N.After the last wash, resuspend the cells in 3 milliliters of MSL minus N medium. Measure the optical density and dilute the cells to an optical density 600 of 1.5. To begin the preparation of agarose pads, first melt an Aliquat of MSL minus N agarose 2 percent in a 95 degree Celsius heat block for 10 to 15 minutes.In the meantime, pellet 100 microliters of the cell suspension prepared earlier at 1000 x g for one minute. Remove the supernatant and resuspend cells in the residual 2 to 4 microliters of medium. With the cells waiting on the bench, add 200 microliters of the melted medium on a glass slide between two spacers.Gently place another slide on top of the melted agar. Once the agarose has solidified, carefully remove the spacers. To remove the top glass slide, gently rotate and pull it sideways.If several strains are to be imaged simultaneously, the pad can be subdivided into four mini pads. For this, use a razor blade and split the pad in its middle pulling the parts about 1 millimeter aside and repeat in the perpendicular direction. Add 1 microliter of cells to the agarose pad and wait for about one minute.Cover the cells with a cover slip and seal the entire periphery of the cover slip with heated VELAP. It is important to seal the entire cover slip to prevent desiccation of the pad. Let the pad sit for at least 30 minutes before imaging.To ensure success of the protocol, and high mating efficiency, cells are to be kept in exponential growth until nitrogen starvation and mounted without excess liquid so they are stationary on the agarose pad. Live cell imaging of the mating yeast cells is then performed at 25 degrees Celsius, or room temperature, following the procedure described in the protocol text. Time-lapse imaging allows a visualization of cells undergoing shmooing, fusion, and sporulation in the 24 hours that follow nitrogen starvation.This process is illustrated in this representative movie by the two cells magnified in the bottom-left inset. This protocol can also be used to monitor the dynamics of fluorescently tagged proteins in mating cells. Here, a heterothallic H-strain expressing myo52 tagged with 3GFP was mated with a heterothallic H+strain expressing myo52 fused to the red fluorescent tdTomato protein, as well as cytosolic GFP.The cytosolic GFP allows monitoring of the timing of fusion, visualized by its entry into the H-cell. These time-lapse images show that myo52 initially formed to dynamic zones throughout the cell cortex. Myo52 signal was then stabilized in a single focus just before the fusion event.After watching this video, you should have a good understanding of how to prepare cells for efficient mating and live cell investigation of the mating process of fission yeast.

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