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>
		<li>Use the following Vitamins (1,000x) stock: Pantothenate: 1 g/L, Nicotinic Acid: 10 g/L, Inositol: 10 g/L, Biotin: 10 mg/L. Filter-sterilize using a 0.22 &#956;m pore size filter and store at 4 &#176;C.</li>
		<li>Use the following Trace elements (10,000x) stock: Boric Acid: 5 g/L, MnSO4: 4 g/L, ZnSO4&#183;7H2O: 4 g/L, FeCl2&#183;6H2O: 2 g/L, MoO3: 0.4 g/L, KI: 1 g/L, CuSO4&#183;5H2O: 0.4 g/L, Citric Acid: 10 g/L. Filter-sterilize using a 0.22 &#956;m pore size filter and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare MSL-N Agarose 2% (used to make agarose pad chambers) by combining 10 ml of MSL-N with 0.2 g of agarose. Melt for ~2 min at high power in a microwave oven until agarose dissolves and aliquot 0.5 ml in microcentrifuge tubes. Store at RT.</li>
	<li>Prepare Minimum Sporulation medium with nitrogen (MSL+N) from MSL-N by adding (NH4)2SO4: 2 g/L, Leucine: 0.225 g/L, Adenine: 0.225 g/L, Uracil: 0.225 g/L. Filter-sterilize using a 0.22 &#181;m pore size filter. Store at RT. 
	Note: Leucine, adenine and uracil are added to this medium to allow the growth of auxotrophic strains. Additional amino-acid supplement should be included if the strain used carries a distinct auxotrophy (for instance his3&#916;). Please also note that work with fully prototroph strains is recommended, as only such strains will allow high mating efficiency.</li>
	<li>Prepare 200 ml VALAP for chamber sealing. In a beaker add equal weights of Lanolin, Vaseline (or other petroleum jelly), and Paraffin. Heat mixture at low temperature and stir occasionally until thoroughly blended. Aliquot the mix into several small petri dishes. Store at RT.</li>
</ol>
2. Culturing Fission Yeast Strains for Mating Experiments (Figure 1).
<ol>
	<li>Day 1, Evening: 
	Inoculate freshly streaked strains from solid media into culture tubes containing 3 ml of MSL+N. Use flat-bottom tubes of about 2.5 cm diameter. Use other culture tubes or flasks with adapted culture volume. Incubate overnight (O/N) with shaking at 25 &#176;C, 200 rpm. If working with heterothallic strains, inoculate separately.
	<ol>
		<li>Dilute cell suspensions in media the following morning to ensure that cultures have optical density measured at 600 nm (O.D.600) of 0.4-0.8 in the evening. 
		Note: O.D.600 measurements as a proxy of cell concentration for fission yeast are linear in the range of approximately 0.1-1.0. For our spectrophotometer O.D.600&#160;= 0.1 corresponds to 1.4 x 106 cells per ml. Thus, if initial O.D.600 reads are outside this range samples need to be diluted/concentrated for reliable measurements.</li>
	</ol>
	</li>
	<li>Day 2, Evening: 
	Dilute cells in 20 ml of MSL+N media to O.D.600 = 0.025 in 100 ml flasks. Incubate strains O/N at 30 &#176;C, 200 rpm. 
	Note: Wild-type cell cultures should reach O.D.600 ~0.8 the following morning (15 hr&#160;later). If working with strains with longer generation time adjust dilution accordingly.</li>
	<li>Day 3, Morning: 
	Measure O.D.600 to verify that cultures have cell density of O.D.600 ~0.8. If working with heterothallic strains, mix equal numbers of partner cells. Pellet cells at 1,000 x g and transfer to a 1.5 ml tube. Wash cells three times in 1 ml of MSL-N medium. Re-suspend cells in 3 ml of MSL-N medium and dilute cells to O.D.600 = 1.5.
	<ol>
		<li>To monitor mating between sister cells directly mount cells for imaging (see Protocol Section 2.4). Use homothallic h90 strains, in which mating type switching will take place during the mitotic divisions occurring after nitrogen deprivation. Immediate mounting of cells on the agarose pad ensures that, after cell division, sister-cells remain next to each other.</li>
		<li>To monitor cell polarization (exploratory dynamics and shmooing) incubate 1-3 ml of cell culture at 30 &#176;C, 200 rpm for 3-4 hr prior to mounting cells for imaging. Within these 3-4 hr, the last mitotic division will have occurred. A few cells will have initiated polarization, but most will just be starting their exploratory polarization dynamics.</li>
		<li>To monitor cell-cell fusion incubate 1-3 ml of cell culture at 30 &#176;C, 200 rpm for 4-6 hr prior to mounting cells for imaging.</li>
		<li>To monitor post-fusion events incubate 1-3 ml of cell cultures at 30 &#176;C, 200 rpm for 8 hr prior to mounting cells for imaging.</li>
		<li>To monitor response to a specific pheromone concentration (only for heterothallic strains) incubate 3 ml of cell culture at 30 &#176;C, 200 rpm for 3-4 hr prior to mounting cells for imaging.
		<ol>
			<li>Remove the MSL-N-containing microcentrifuge tube from the 95 &#176;C heat block (see Protocol section 2.4.1. below). Add P-factor or M-factor at the desired concentration directly in the melted MSL-N agarose. Mix well by vortexing before immediate pad preparation.</li>
			<li>After cell mounting, incubate the pad for 15-30 min at 25 &#176;C prior to imaging. P-factor and M-factor pheromones were used from a stock solution of 1 mg/ml in methanol. 
			Note: If working with mutant strains incubation times may vary. Clumping of cells may occur after prolonged incubation in liquid media and may be particularly strong in some mutant strains. Clumped cells cannot be spotted onto agarose pads in a single layer and thus are difficult to autofocus and image. In case of extensive cell clumping mount cells on agarose pads immediately after washes and re-suspension in nitrogen-lacking media (as in the Protocol Section 2.3.1.) and incubate them at 30 &#176;C prior to imaging.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Mounting Cells for Imaging:
	<ol>
		<li>Prepare MSL-N agarose pads16 by melting an aliquot in a 95 &#176;C heat-block for 10-15 min and adding 200 &#956;l between two glass slides separated by spacers (Figure 1B). Use spacers with a thickness of ~0.5 mm. To make spacers, use strips cut out of cardboard, such as that delivered with restriction enzymes.</li>
		<li>Pellet 100 &#956;l of cells at 1,000 x g for 1 min, remove the supernatant and re-suspend cells in residual 2-4 &#956;l medium. Do NOT re-suspend in fresh medium as it delays mating.</li>
		<li>After 2-3 min carefully remove spacers and the top glass slide.&#160;If several strains are to be mounted, prepare the cell concentrates before preparing the agarose pad.</li>
		<li>Add 1 &#956;l of cells to the pad and wait until the drop starts drying (~1 min) before covering with cover slip and sealing with VALAP.</li>
		<li>Let the pad sit for at least 30 min before imaging at 25 &#176;C or RT.</li>
		<li>To obtain a mixture of cells in various stages of mating, make a pad immediately after washes in MSL-N (see Section 2.3.) and incubate at 18 &#176;C O/N (~15 hr). This approach is useful for imaging cells at distinct mating stages with high temporal resolution or samples with weak signal.</li>
	</ol>
	</li>
</ol>
3. Live-cell Imaging of Mating Yeast Cells
<ol>
	<li>Adjust Image Acquisition Settings. 
	Note: Image acquisition settings presented here were optimized for the DeltaVision platform composed of a customized Olympus IX-71 inverted microscope with Plan Apo 60X/1.42 NA or U-Plan Apo 100X/1.4 NA objectives, a CoolSNAP HQ2 camera and an Insight SSI 7 color combined unit illuminator. The hardware is controlled by softWoRx v4.1.2 that also enables digital autofocusing.
	<ol>
		<li>Ensure that the autofocusing intervals do not exceed 15 min since the Z-drift over longer time can surpass the capacity of the software autofocusing system. If using a hardware-based autofocusing system, use larger intervals.</li>
		<li>Adjust imaging interval. Take images every 10 min for ~15 hr&#160;as a starting point when working with a fluorescently tagged protein for the first time. This interval provides a good overview of the entire mating process without extensive bleaching.
		<ol>
			<li>Image with 5 min intervals or shorter to more precisely time events such as fusion. Shorter intervals require strong fluorophores enabling low exposure times and little bleaching. For imaging with intervals below 1 min avoid O/N microscopy and instead prepare a mixture of all mating stages as detailed in Protocol Section 2.4.6.</li>
		</ol>
		</li>
		<li>Adjust image exposure time. Test exposure times between 50 and 300 msec. Aim to strike a balance between the signal-to-noise ratio and photobleaching as to achieve a large number of time-points with a discernable signal (typically over 100).</li>
		<li>Adjust Z-sectioning. Sections every 0.3 &#181;m covering 5 &#181;m provide a good imaging depth. Note that Z-sectioning further increases photo-damage, which can be minimized by using the OAI (optical axis integration - a single sweep acquisition of the total sample depth). Good auto-focus system reduces the need for many Z-sections and is highly recommended.</li>
	</ol>
	</li>
	<li>Tips to Study Mating Type-specific Behaviors:
	<ol>
		<li>For heterothallic strains, use h- and h+ cells expressing distinct fluorophores, so that the two cell types can be distinguished. Use any genetically encoded fluorophore expressed as a cytosolic version or fusion to an endogenous protein, though we have had good success with sfGFP, a fast-folding variant of GFP17. Endogenous proteins are usually tagged at their endogenous genomic locus by homologous recombination14.</li>
		<li>For homothallic strains, express a genetically encoded cytosolic fluorescent protein under the control of mating type specific promoters such as those of the pheromone receptor genes map3 and mam2 to drive the expression in P and M cells, respectively. The promoters of map3 and mam2 are inactive during vegetative growth and are induced upon nitrogen starvation18,19. Constructs driving the expression of cytosolic GFP or mCherry under the control of these promoters are typically integrated at a genomic locus (ura4, leu1) that does not interfere with the normal progression of the sexual life cycle12.</li>
	</ol>
	</li>
	<li>Use mating type specific cytosolic fluorophores (as in Protocol Section 3.2.2) to determine precise timing of cell fusion visualized as the transfer of fluorescent signal from one partner cell to the other.</li>
</ol>
4. Quantification of Mating and Fusion Efficiencies
<ol>
	<li>To quantify mating and fusion efficiencies11,12, spot cells directly after MSL-N wash onto MSL-N agarose pads, as in step 2.3.1 above, and leave for 24 hr at 25 &#176;C prior to imaging (Figure 1A). Using this protocol a homothallic wild-type strain mating efficiency reaches ~60% and fusion efficiency 99%. To test whether any reduction in these values observed in mutant strains reflect a terminal phenotype or a delay, repeat the experiment with a 36 hr incubation time.</li>
	<li>Calculate mating efficiency as <img alt="Equation1" src="/files/ftp_upload/53801/53801eq1.jpg" /> Mating pairs include zygotes, asci, and unfused cell pairs identified from their position and growth towards each others.</li>
	<li>Calculate fusion efficiency as <img alt="Equation2" src="/files/ftp_upload/53801/53801eq2.jpg" /> 
	Note: Fused mating pairs are identified by their ability to form spores if it is known that the mutant under study does not influence sporulation. If sporulation cannot be used as read-out, fusion is determined by observing the spreading of a cytosolic marker expressed in only one mating partner into both partners.</li>
</ol>

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The authors declare that they have no competing financial interests.

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.

<table><tbody><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-10KG</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>1.05108.0050</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71381</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>63140</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>12095</td><td></td></tr><tr><td>Pantothenate</td><td>AppliChem</td><td>A2088,0025</td><td></td></tr><tr><td>Nicotinic Acid</td><td>AppliChem</td><td>A0963,0100</td><td></td></tr><tr><td>Inositol</td><td>AppliChem</td><td>A1716,0100</td><td></td></tr><tr><td>Biotin</td><td>AppliChem</td><td>A0967,0250</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B6768-1KG</td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A1038,0250</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Z4750</td><td></td></tr><tr><td>FeCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>AppliChem</td><td>A3514,0250</td><td></td></tr><tr><td>Molybdenum oxide (VI) (MoO&lt;sub&gt;3)&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>69850</td><td></td></tr><tr><td>KI</td><td>AppliChem</td><td>A3872,0100</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>AppliChem</td><td>A1034,0500</td><td></td></tr><tr><td>Citric Acid&amp;nbsp;</td><td>AppliChem</td><td>A2344,0500</td><td></td></tr><tr><td>Agarose</td><td>Promega</td><td>V3125</td><td></td></tr><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Merck</td><td>1.01217.1000</td><td></td></tr><tr><td>L-Leucine</td><td>Sigma-Aldrich</td><td>L8000-100G</td><td></td></tr><tr><td>Adenine Hemisulfat Salt, mini 99%</td><td>Sigma-Aldrich</td><td>A9126-100G</td><td></td></tr><tr><td>Uracil&amp;nbsp;</td><td>Sigma-Aldrich</td><td>U0750</td><td></td></tr><tr><td>Lanolin</td><td>Sigma-Aldrich</td><td>L7387</td><td></td></tr><tr><td>Vaseline</td><td>Reactolab</td><td>92045-74-4</td><td></td></tr><tr><td>Paraffin</td><td>Reactolab</td><td>7005600</td><td></td></tr></tbody></table>

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...

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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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14.5K Views.  University of Lausanne. 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. 

Video: Microscopy of Fission Yeast Sexual Lifecycle

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Genetics

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

Biochemistry

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

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.

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.

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

Identifying Drugs that Affect Polymerization of Bacterial Cytoskeletal Proteins

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB ...

Microscopy of Fission Yeast Sexual Lifecycle

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