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.

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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...

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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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14.4K 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

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

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 ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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 ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

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 ...

Microscopy of Fission Yeast Sexual Lifecycle

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Purification of Mitochondria from Yeast Cells

Yeast Colony Embedding Method

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

High-throughput Yeast Plasmid Overexpression Screen

Competitive Genomic Screens of Barcoded Yeast Libraries

Chromatographic Purification of Highly Active Yeast Ribosomes

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Sexual Development and Ascospore Discharge in Fusarium graminearum

Surface Spreading and Immunostaining of Yeast Chromosomes

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast