This work was supported by the Biotechnology and Biological Sciences Research Council (grant 31/C15982), the Canadian Institutes of Health Research, the Canada Foundation for Innovation, the British Columbia Knowledge Development Fund, the Michael Smith Foundation for Health Research (grant to C.J.R. Loewen), and Fight For Sight.

Materials and methods:
<ol>
<li>Yeast strains: 
<ul>
<li>Acp1-GFP (GFP::His): C-terminal GFP-tagging of Acp1 was generated by a PCR-mediated homologous recombination using plasmid pKT128 (contains GFP and HIS5). Positive transformants were confirmed by confocal microscopy and colony PCR. The strain background was BY7043 (MAT alpha can1&#x2206;::STE2pr-lue2 lyp1&#x2206; his3&#x2206;1 leu2&#x2206;0 ura3&#x2206;0 met15&#x2206;0) from Boone lab (Tong and Boone, 2006).</li>
<li>Deletion Mutant Array (DMA) (xxx::KanR): it is an array of about 5,000 deletion mutants of non-essential genes. All of these nonessential genes were knocked out on G418. The strain background of the array is BY4741 (MATa his3_1 leu2_0 met15_0 ura3_0). Our DMA was originally from Boone lab.</li>
</ul>
</li>
<li>Incorporating Acp1-GFP in the DMA:
 The following protocol for making a GFP array is adapted from the Synthetic Genetic Analysis (SGA) technology (Tong and Boone, 2006) with modifications. All of the replica pinning steps are done by using a Singer ROTOR high-density arraying robotic system. Alternatively, manual pinning or a liquid-based high-density arraying robot may be used. Acronyms: YPD, yeast extract-peptone-dextrose; SD, synthetic dropout; LRK, Lue/Arg/Lys dropout media; LHRK, Lue/His/Arg/Lys dropout media Preparation
<ol>
<li>Grow a lawn of Acp1-GFP cells by pouring a 5 ml of overnight Acp1-GFP culture onto a YPD plate. Allow the plate to dry sufficiently by a flame or in a biological fume hood.</li>
<li>Incubate the plate at 30&#x02DA;C overnight.</li>
<li>Using a robot, array Acp1-GFP strain into 1536 colonies per plate. At the same time, prepare a fresh copy of the DMA by replica pinning to YPD + G418 plates.</li>
<li>Incubate the plates at 30&#x02DA;C overnight. 
Mating and diploid selection </li>
<li>Mate the Acp1-GFP strain with the DMA by replica pinning and over-laying the colonies on YPD plates.</li>
<li>Incubate the plates at 30&#x02DA;C overnight.</li>
<li>Replica-plate the colonies onto SD &ndash; His + G418 plates to select diploid cells (MATa/MATalpha).</li>
<li>Incubate the plates at 30&#x02DA;C overnight. 
Sporulationg and germination </li>
<li>Replica-plate the diploid colonies on enhanced sporulation media, which contains reduced levels of carbon and nitrogen sources to induce the formation of meiotic spores.</li>
<li>Incubate the plates at 25&#x02DA;C for a minimum of 5 days.</li>
<li>Germinate the MATa meiotic progenies by replica-plating the colonies onto LRK media and incubate the plates at 30&#x02DA;C for two days.</li>
<li>This media will induce the germination of haploid cells from spores. Since a LUE2 opening reading frame (ORF) is integrated at the MATa-specific STE2 promoter, all the haploids germinated will be MATa. 
Selection of both alleles </li>
<li>It is now necessary to select both the GFP allele and the single mutant allele. To accomplish this, MATa cells are replica-plated onto LRK + G418 media, which selects for haploid cells that carry the gene deletion (xxx::kanR).</li>
<li>Incubate the plates at 30&#x02DA;C overnight.</li>
<li>Finally, the MATa meiotic progenies are replica-plated onto LHRK + G418 media to select for growth of single mutants (xxx::kanR) also harboring Acp1-GFP (GFP::His). These plates can be stored at 4&#x02DA;C for up to 3 months.</li>
</ol>
 The following protocol for making a GFP array is adapted from the Synthetic Genetic Analysis (SGA) technology (Tong and Boone, 2006) with modifications. All of the replica pinning steps are done by using a Singer ROTOR high-density arraying robotic system. Alternatively, manual pinning or a liquid-based high-density arraying robot may be used. Acronyms: YPD, yeast extract-peptone-dextrose; SD, synthetic dropout; LRK, Lue/Arg/Lys dropout media; LHRK, Lue/His/Arg/Lys dropout media Preparation
</li>
<li>Microscopy:<ol>
<li>Pick the desired mutant(s) expressing Acp1-GFP directly from plate.</li>
<li>Grow cells at 30 &deg;C in YPD or SD &ndash; His media to early log phase.</li>
<li>Visualize by confocal microscopy using the GFP filterset. An example of expected result is show in Figure 1.</li>
</ol></li>
</ol>
SGA media:
The growth media (YPD) and selecting media (SD) used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions.
LRK, LHRK media (for 400 ml total)
<table border="0">
<tbody>
<tr>
<td>Add in order</td>
<td>Amount</td>
</tr>
<tr>
<td>Yeast nitrogenous base with ammonium sulfate</td>
<td>2.7 g</td>
</tr>
<tr>
<td>Amino acid mix</td>
<td>0.8 g</td>
</tr>
<tr>
<td>Agar</td>
<td>8 g</td>
</tr>
<tr>
<td>dH2O</td>
<td>360 mL</td>
</tr>
<tr>
<td colspan="2">Autoclave</td>
</tr>
<tr>
<td>20 % Dextrose</td>
<td>40 ml</td>
</tr>
<tr>
<td colspan="2">Cool to 65&#x02DA;C</td>
</tr>
<tr>
<td>100 mg/ml canavanine</td>
<td>0.2 ml</td>
</tr>
<tr>
<td>100 mg/ml thialysine</td>
<td>0.2 ml</td>
</tr>
<tr>
<td colspan="2">Mix, pour 50 ml per plate</td>
</tr>
</tbody>
</table>
LRK, LHRK + G418 media (for 400 ml total)
<table border="0">
<tbody>
<tr>
<td>Add in order</td>
<td>Amount</td>
</tr>
<tr>
<td>Yeast nitrogenous base without ammonium sulfate</td>
<td>0.7 g</td>
</tr>
<tr>
<td>Amino acid mix</td>
<td>0.8 g</td>
</tr>
<tr>
<td>Monosodium Glutamate</td>
<td>0.4</td>
</tr>
<tr>
<td>Agar</td>
<td>8 g</td>
</tr>
<tr>
<td>dH2O</td>
<td>360 mL</td>
</tr>
<tr>
<td colspan="2">Autoclave</td>
</tr>
<tr>
<td>20 % Dextrose</td>
<td>40 ml</td>
</tr>
<tr>
<td colspan="2">Cool to 65&#x02DA;C</td>
</tr>
<tr>
<td>100 mg/ml canavanine</td>
<td>0.2 ml</td>
</tr>
<tr>
<td>100 mg/ml thialysine</td>
<td>0.2 ml</td>
</tr>
<tr>
<td>200 mg/ml G418</td>
<td>0.4 ml</td>
</tr>
<tr>
<td colspan="2">Mix, pour 50 ml per plate</td>
</tr>
</tbody>
</table>
Enriched sporulation media (for 400 ml total)
<table border="0">
<tbody>
<tr>
<td>Add in order</td>
<td>Amount</td>
</tr>
<tr>
<td>Potassium acetate</td>
<td>4 g</td>
</tr>
<tr>
<td>Yeast extract</td>
<td>0.4 g</td>
</tr>
<tr>
<td>Dextrose</td>
<td>0.2 g</td>
</tr>
<tr>
<td>Sporulation amino acid mix</td>
<td>0.04 g</td>
</tr>
<tr>
<td>Agar</td>
<td>8 g</td>
</tr>
<tr>
<td>dH2O</td>
<td>360 mL</td>
</tr>
<tr>
<td colspan="2">Autoclave</td>
</tr>
<tr>
<td colspan="2">Cool to 65&#x02DA;C</td>
</tr>
<tr>
<td>200 mg/ml G418</td>
<td>0.4 ml</td>
</tr>
<tr>
<td colspan="2">Mix, pour 50 ml per plate</td>
</tr>
</tbody>
</table>

<ol>
	<li>Amberg, D. C., Burke, D., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual. , (2005).</li><li>Tong, A. H., Boone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+genetic+array+analysis+in+Saccharomyces+cerevisiae.">Synthetic genetic array analysis in Saccharomyces cerevisiae.</a> Methods Mol Biol. 313, 171-192 (2006).</li></ol>

This method can help efficiently incorporate a mitochondrial marker, Acp1-GFP into various mutant backgrounds. It relies on the use of a robotic system, and can easily adopted for use with any robotic system.&nbsp;This procedure can be also used for incorporating other types of markers. For example, to visualize ER, we routinely use the marker Erg11-GFP. In our representative images, a mutant and a wild type with the Acp1-GFP maker were visualiz ed by confocal microscopy to study the mitochondria morphology. The mutant s...

<table><tbody><tr><td>SGA media</td><td></td><td></td><td>The growth media (YPD) and synthetic dropout media (SD) including LRK and LHRK, and enriched sporulation media used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions.</td></tr></tbody></table>

a high-throughput method to globally study the organelle morphology in s. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

Watch this Scientific Journal Video about A high-throughput method to globally study the organelle morphology in S. cerevisiae at JoVE.com

A high-throughput method to globally study the organelle morphology in S. cerevisiae

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

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JoVE Journal

Biology

10.3K Views.  University of British Columbia - UBC. GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Video: A high-throughput method to globally study the organelle morphology in S. cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Biochemistry

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 ...

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 ...

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 ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Bioengineering

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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

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

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

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 ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

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 ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

Summary

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Chapters in this video

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Visualizing Yeast Organelles with Fluorescent Protein Markers

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

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

4D Microscopy of Yeast

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

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae