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>
		...

<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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+and+fuse:+how+yeast+cells+do+it.">Mate and fuse: how yeast cells do it.</a> Open Biol. 3 (3), 130008 (2013).</li><li>Mochizuki, N., 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=Reduction+in+the+intracellular+cAMP+level+triggers+initiation+of+sexual+development+in+fission+yeast.">Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast.</a> Mol Gen Genet. 233 (1-2), 17-24 (1992).</li><li>Sugimoto, A., Iino, Y., Maeda, T., Watanabe, 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+ste11++encodes+a+transcription+factor+with+an+HMG+motif+that+is+a+critical+regulator+of+sexual+development.">Schizosaccharomyces pombe ste11+ encodes a transcription factor with an HMG motif that is a critical regulator of sexual development.</a> Genes Dev. 5 (11), 1990-1999 (1991).</li><li>Kjaerulff, S., Lautrup-Larsen, I., Truelsen, S., Pedersen, M., 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=Constitutive+activation+of+the+fission+yeast+pheromone-responsive+pathway+induces+ectopic+meiosis+and+reveals+ste11+as+a+mitogen-activated+protein+kinase+target.">Constitutive activation of the fission yeast pheromone-responsive pathway induces ectopic meiosis and reveals ste11 as a mitogen-activated protein kinase target.</a> Mol Cell Biol. 25 (5), 2045-2059 (2005).</li><li>Obara, T., Nakafuku, M., Yamamoto, M., Kaziro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+gene+encoding+a+G-protein+alpha+subunit+from+Schizosaccharomyces+pombe:+involvement+in+mating+and+sporulation+pathways.">Isolation and characterization of a gene encoding a G-protein alpha subunit from Schizosaccharomyces pombe: involvement in mating and sporulation pathways.</a> Proc Natl Acad Sci U S A. 88 (13), 5877-5881 (1991).</li><li>Toda, 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=Fission+yeast+genes+that+confer+resistance+to+staurosporine+encode+an+AP-1-like+transcription+factor+and+a+protein+kinase+related+to+the+mammalian+ERK1/MAP2+and+budding+yeast+FUS3+and+KSS1+kinases.">Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases.</a> Genes Dev. 5 (1), 60-73 (1991).</li><li>Bendezu, F. 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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		<tr height="21">
			<td height="21" style="height:21px;">MgSO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>63140</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>12095</td>
			<td></td>
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			<td height="17" style="height:17px;">Pantothenate</td>
			<td>AppliChem</td>
			<td>A2088,0025</td>
			<td></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 height="17" style="height:17px;">Biotin</td>
			<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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		<tr height="21">
			<td height="21" style="height:21px;">MnSO4</td>
			<td>AppliChem</td>
			<td>A1038,0250</td>
			<td></td>
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			<td height="21" style="height:21px;">ZnSO4.7H2O&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>Z4750</td>
			<td></td>
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			<td height="21" style="height:21px;">FeCl2.6H2O</td>
			<td>AppliChem</td>
			<td>A3514,0250</td>
			<td></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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		<tr height="17">
			<td height="17" style="height:17px;">KI</td>
			<td>AppliChem</td>
			<td>A3872,0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<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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		<tr height="17">
			<td height="17" style="height:17px;">Agarose</td>
			<td>Promega</td>
			<td>V3125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(NH4)2SO4&#160;</td>
			<td>Merck</td>
			<td>1.01217.1000</td>
			<td></td>
		</tr>
		<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>
		<tr height="17">
			<td height="17" style="height:17px;">Lanolin</td>
			<td>Sigma-Aldrich</td>
			<td>L7387</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vaseline</td>
			<td>Reactolab</td>
			<td>92045-74-4</td>
			<td></td>
		</tr>
		<tr height="17">
			<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 ...

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