Name: Temporal Quantification of MAPK Induced Expression in Single Yeast Cells (Video) | JoVE Journal
Uploaded: 2013-10-04T23:00:00.000+00:00
Duration: 7 min 59 s
Description: Learn temporal quantification of MAPK induced expression in single yeast cells using live cell microscopy and flow cytometry. This method enables precise measurement of gene expression dynamics in Saccharomyces cerevisiae.

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5 minutes

The authors thank Matthias Peter and his group at the Institute of Biochemistry at the ETH in Z&uuml;rich where these methods have been developed. This work has been supported by the Swiss National Science Foundation.

1. Microscopy Measurements
<ol>
	<li>Inoculate 5 ml of synthetic medium with yeast.</li>
	<li>Grow cells at 30 &#176;C overnight.</li>
	<li>Measure the OD600 of the overnight culture the next morning.</li>
	<li>Dilute overnight culture in 5 ml synthetic medium to OD600 0.05.</li>
	<li>Grow the diluted culture at 30 &#176;C for at least 4 hr.</li>
	<li>Prepare 3-fold concentrated (0.6 M) NaCl solution in synthetic medium.</li>
	<li>Prepare a 1 mg/ml solution of Concanavalin A (ConA) in PBS.</li>
	<li>Filtrate ~200 &#956;l of ConA solution into the well slide.</li>
	<li>Let stand for 30 min.</li>
	<li>Remove ConA solution.</li>
	<li>Add 150 &#956;l H2O to the well.</li>
	<li>Remove H2O.</li>
	<li>Measure OD600 of cell culture.</li>
	<li>Prepare 300 &#956;l of cell culture at OD600 = 0.02 in a 1.5 ml microtube.</li>
	<li>Sonicate the cells in a water bath for 45 sec.</li>
	<li>Vortex briefly the microtube.</li>
	<li>Sonicate the cells in a water bath for 45 sec.</li>
	<li>Vortex briefly the microtube.</li>
	<li>Add 200 &#956;l of cell culture to the well.</li>
	<li>Wait 30 min to let the cells settle to the bottom of the well.</li>
	<li>Place the well slide on the microscope.</li>
	<li>Select the illumination settings for the fluorescent channel to be recorded.</li>
	<li>Select the illumination settings for two bright field images (one slightly out of focus: 3 &#956;m below the focal plane to allow for a proper segmentation of the cells).</li>
	<li>Select the time intervals for the time-lapse measurement</li>
	<li>Select the fields of view to image.</li>
	<li>Start the acquisition of a few frames.</li>
	<li>Pause the acquisition to add the 100 &#956;l of 0.6 M NaCl medium.</li>
	<li>Resume imaging.</li>
</ol>
2. Flow Cytometry Measurements
<ol>
	<li>Inoculate 5 ml of synthetic medium with yeast.</li>
	<li>Grow at 30 &#176;C overnight.</li>
	<li>Measure the OD600 of the overnight culture the next morning.</li>
	<li>Dilute overnight culture in 5 ml synthetic medium to OD600 0.1.</li>
	<li>Grow at 30 &#176;C for at least 4 hr to reach an OD600 of 0.2-0.4.</li>
	<li>Prepare cycloheximide solution 1 mg/ml in H2O.</li>
	<li>Prepare 3-fold concentrated (0.6 M) NaCl solution in synthetic medium.</li>
	<li>Prepare one 1.5 ml microtube with 100 &#956;l of 0.6 M NaCl medium for each time point to be measured.</li>
	<li>At time 0, add 200 &#956;l of cell culture to each microtube.</li>
	<li>Incubate with shaking at 30 &#176;C.</li>
	<li>At time T, add 30 &#956;l cycloheximide to a microtube.</li>
	<li>Repeat step 2.11 for all the desired time points.</li>
	<li>Incubate all microtubes for at least 45 min and up to 3 hr at 30 &#176;C with shaking.</li>
	<li>Prepare FACS tubes with 400 &#956;l PBS.</li>
	<li>Sonicate the cells in water bath for 1 min.</li>
	<li>Vortex briefly the microtubes.</li>
	<li>Sonicate the cells in water bath for 1 min.</li>
	<li>Vortex briefly the microtubes.</li>
	<li>Add 100 &#956;l cell culture from each microtube to its corresponding FACS tube.</li>
	<li>Select the 488 nm excitation laser and 530/30 nm bandpass filter for detection.</li>
	<li>Test non-expressing and fully expressing sample to verify if they fall in the detector sensitivity range.</li>
	<li>Measure 10,000 cells for each sample acquired.</li>
</ol>

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<li>de Nadal, E., Zapater, M., Alepuz, P. M., Sumoy, L., Mas, G., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+MAPK+Hog1+recruits+Rpd3+histone+deacetylase+to+activate+osmoresponsive+genes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes.</a> Nature. 427 (6972), 370-374 (2004).</li>
<li>Ghaemmaghami, S., Huh, W. -K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+analysis+of+protein+expression+in+yeast%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li>
<li>Wu, J. -Q., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Counting+cytokinesis+proteins+globally+and+locally+in+fission+yeast%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Counting cytokinesis proteins globally and locally in fission yeast.</a> Science. 310 (5746), 310-314 (2005).</li>
<li>Colman-Lerner, A., Gordon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulated+cell-to-cell+variation+in+a+cell-fate+decision+system%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Regulated cell-to-cell variation in a cell-fate decision system.</a> Nature. 437 (7059), 699-706 (2005).</li>
<li>Acar, M., Becskei, A., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancement+of+cellular+memory+by+reducing+stochastic+transitions%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Enhancement of cellular memory by reducing stochastic transitions.</a> Nature. 435 (7039), 228-232 (2005).</li>
<li>Pelet, S., Rudolf, F., Nadal-Ribelles, M., de Nadal, E., Posas, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+activation+of+the+HOG+MAPK+pathway+regulates+bimodal+gene+expression%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Transient activation of the HOG MAPK pathway regulates bimodal gene expression.</a> Science. 332 (6030), 732-735 (2011).</li>
<li>de Nadal, E., Casadome, L., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+MEF2-like+transcription+factor+Smp1+by+the+stress-activated+Hog1+mitogen-activated+protein+kinase%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">Targeting the MEF2-like transcription factor Smp1 by the stress-activated Hog1 mitogen-activated protein kinase.</a> Molecular and Cellular Biology. 23 (1), 229-237 (2003).</li>
<li>Elowitz, M. B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stochastic+gene+expression+in+a+single+cell%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li>
<li>Pelet, S., Dechant, R., Lee, S. S., van Drogen, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+integrated+image+analysis+platform+to+quantify+signal+transduction+in+single+cells%5BTitle%5D)+AND+%22Integrative+biology+%3A+quantitative+biosciences+from+nano+to+macro%22%5BJournal%5D)">An integrated image analysis platform to quantify signal transduction in single cells.</a> Integrative biology : quantitative biosciences from nano to macro. 4 (10), 1274-1282 (2012).</li>
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<li>Chernomoretz, A., Bush, A., Yu, R., Gordon, A., Colman-Lerner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+Cell-ID+1.4+with+R+for+microscope-based+cytometry%5BTitle%5D)+AND+%22Current+protocols+in+molecular+biology%22%5BJournal%5D)">Using Cell-ID 1.4 with R for microscope-based cytometry.</a> Current protocols in molecular biology. Chapter 14, Unit 14.18(2008).</li>
<li>Dimopoulos, S., Mayer, C., Rudolf, F., Stelling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automatic+Single+Cell+Segmentation+in+a+Variety+of+Microscopy+Images%5BTitle%5D)+AND+%22Current+protocols+in+molecular+biology%22%5BJournal%5D)">Automatic Single Cell Segmentation in a Variety of Microscopy Images.</a> Current protocols in molecular biology. , (2012).</li>
<li>Miyawaki, A., Nagai, T., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+protein+fluorophore+formation+and+engineering%5BTitle%5D)+AND+%22Current+Opinion+in+Chemical+Biology%22%5BJournal%5D)">Mechanisms of protein fluorophore formation and engineering.</a> Current Opinion in Chemical Biology. 7 (5), 557-562 (2003).</li>
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The authors declare that they have no competing financial interests.

Microscopy
The treatment of the well with ConA is an essential step to ensure a proper imaging of the cells. Because ConA has low solubility in PBS (5 mg/ml), the filtration process allows the removal of large aggregates that are present in the unfiltered solution and interfere with the imaging. Cells attach relatively strongly to the treated surface and the addition of the inducing solution should not disturb the localization of the cells, allowing for a continuous tracking before and after the ...

Signaling via transduction cascades often culminates in the expression of proteins. The characterization of this expression profile is a key element in understanding the function of biological pathways. The identification of the spectrum of up-regulated proteins and the dynamics of their activation can be achieved by various techniques such as micro-arrays, northern blots or western blots1-3. However, these techniques average the response of an entire population of cells. To understand the fine regulation of the expression of proteins, it is desirable to gather measurements at the single cell level. Ideally, these measurements should also provide quantitative data amenable to develop mathematical models of the underlying pathway.
Microscopy and flow cytometry are two techniques, which are ideally suited to deliver such quantitative single cell measurements. The endogenous tagging of proteins with a fluorescent protein can be used to quantify their expression level 4. However, since the addition of a large fluorescent moiety at the terminus of the protein can render it non-functional, it is often more desirable to generate specific expression reporters based on a promoter driving the expression of a fluorescent construct. This reporter protein is exogenous to the cellular system and therefore does not influence the signaling events that are taking place in the cell.
Both microscopy and flow cytometry have been widely used in the yeast signaling field. As examples; Colman-Lerner and co-workers correlated, in single cells, the expression of mating specific and constitutively expressed reporters by microscopy to quantify the noise in yeast mating signal transduction cascade5, Acar and colleagues used flow cytometry to study the regulatory network controlling the expression of the GAL genes6. In a previous study7, we have used a combination of these two techniques to study the expression output from the high osmolarity glycerol (HOG) pathway in budding yeast. This mitogen activated protein kinase (MAPK) pathway is triggered by hyper-osmotic stress. It results in the activation of the MAPK Hog1, which translocates to the nucleus of the cell to induce a transcriptional program resulting in the expression of roughly 300 genes. To study this process, we had engineered an expression reporter based on the STL1 promoter (a gene induced specifically in response to Hog1 activity8) driving the expression of a quadruple Venus fluorescent protein (pSTL1-qV). Flow cytometry measurements uncovered the presence of two populations of cells at intermediate stress level (0.1 M NaCl) with only a fraction of the population expressing the fluorescent reporter. We used microscopy to further investigate this behavior and discovered that this noise in protein expression was governed by intrinsic factors9. We could further observe that cells with a similar level of Hog1 activity could display strikingly different expression outcomes. The combination of these two techniques allowed us to demonstrate how the slow remodeling of stress response genes influenced the expression outcome at the single cell level7.
In this paper, we use the expression of the pSTL1-qV reporter induced by hyper-osmotic shock as an example of the quantification of protein expression by microscopy and flow cytometry. The same strain subjected to 0.2 M NaCl stress was studied with both techniques. This will allow us to highlight some key differences in these two highly complementary techniques.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Yeast Nitrogen Base</td><td>ForMedium</td><td>CYN6202</td><td></td></tr><tr><td>CSM amino-acid mix</td><td>ForMedium</td><td>DCS0011</td><td></td></tr><tr><td>Concanavalin A</td><td>GE Healthcare</td><td>17-0450-01</td><td></td></tr><tr><td>Cycloheximide</td><td>Fluka Aldrich Sigma</td><td>C7698</td><td>Toxic</td></tr><tr><td>ySP9</td><td></td><td></td><td>W303Mata leu2::LEU2-pSTL1-quadrupleV enus</td></tr><tr><td>pSP34</td><td></td><td></td><td>pRS305 pSTL1 (-800 -0) - quadruple V enus</td></tr><tr><td colspan="4">&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Ti-Eclipse</td><td></td></tr><tr><td>Microscope control software</td><td>Micro-manager</td><td>Ver 1.4.11</td><td></td></tr><tr><td>Incubation chamber</td><td>LIS</td><td>The Box</td><td></td></tr><tr><td>Fluorescence light source</td><td>Lumencor</td><td>SpectraX</td><td></td></tr><tr><td>Camera</td><td>Hamamatsu</td><td>Flash 4.0</td><td></td></tr><tr><td>Flow cytometer</td><td>BD</td><td>FACS calibur</td><td></td></tr><tr><td>Sonicator bath</td><td>Telesonic</td><td>TUC-150</td><td></td></tr><tr><td>8-well-slide </td><td>Thermo Lab-tek</td><td>155409</td><td></td></tr><tr><td>96-well-plate</td><td>Matrical bioscience</td><td>MGB096-1-2-LG</td><td></td></tr><tr><td>FACS tube</td><td>BD Falcon</td><td>352054</td><td></td></tr></tbody></table>

temporal quantification of mapk induced expression in single yeast cells

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Microscopy
Yeast cells bearing the expression reporter pSTL1-qV (ySP97) were attached to the bottom of the well-slide and placed under the microscope. The cells were stimulated by the addition of stress medium directly into the well during the course of the imaging session. This allows us to acquire a few images of the cells before pathway induction and follow their fate after the stimulation. In the present case, the cells were followed for ~2 hr with a time interval of 10 mi...

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

The overall goal of the following experiment is to quantify mitogen activated protein expression at the single cell level. To achieve this, a yeast strain bearing the fluorescent expression reporter quadruple Venus controlled by the STL one promoter and specific to the activation of the hog one map kinase was generated. Fluorescent protein expression can be quantified either by a live cell microscopy assay in which the cells are stressed directly on the microscope to facilitate real-time apparition of the fluorescence or by flow cytometry, in which case the cells are stressed in a micro tube and the protein synthesis is blocked at a given time.By the addition of cyclo heide, only a few cells at a time can be analyzed with live cell microscopy, but the fate of the individual cells can be directly observed and correlated with other cellular property flow cytometry on the other hand, also allows assessment of mitogen activated protein expression on large numbers of cells at once. This method can help answer key questions in the signaling field, such as which proteins are involved in the regulation of gene expression. Though this method can provide insight into the yeast pathway, it can also be applied to other signaling cascade depending of the availability of the proper expression.Reporter.To prepare the cells for live cell microscopy, begin by inoculating five milliliters of synthetic medium with yeast and then grow the cells at 30 degrees Celsius overnight. The next morning, measure the OD 600 of the overnight culture and then dilute the overnight culture in five milliliters of synthetic medium to an OD 600 of 0.05. Grow the diluted culture at 30 degrees Celsius for at least four more hours.Next, filter about 200 microliters of freshly prepared conna valent a solution into the well slide. After 30 minutes, remove the con a solution and add 150 microliters of water to the well. Now remove the water and let the well dry while the cells are prepared.Measure the OD 600 of the cell culture again, and then dilute the cells to obtain 300 microliters of cell culture at an OD 600 of 0.02 and a 1.5 milliliter micro tube. Sonicate the cells in a water bath for 45 seconds. Briefly vortex the micro tubes and then sonicate and vortex the cells again.Now add 200 microliters of the cells to the well slide, and then after letting the cell settle to the bottom of the well for about 30 minutes, place the slide onto the microscope. Next, select the illumination settings for their fluorescent channel to be recorded and for two bright field images, adjusting one of the bright field settings approximately three microns below the focal plane to allow for proper cell segmentation. Then set the time intervals for the time lapse measurements and select the fields of view for imaging.Now start the acquisition for a few frames and then pause the acquisition to add 100 microliters of freshly prepared 0.6 molar sodium chloride medium, and resume the imaging to prepare the cells for measurement by flow cytometry. Begin by inoculating the yeast in five milliliters of synthetic medium and grow the cells overnight at 30 degrees Celsius. The next morning, measure the OD 600 of the overnight culture and then dilute the cell suspension in five milliliters of synthetic medium to an OD 600 of 0.1.Grow the culture for at least four hours at 30 degrees Celsius to reach an OD 600 of 0.2 to 0.4. Then add 100 microliters of freshly prepared 0.6 molar sodium chloride to a 1.5 milliliter micro tube for each time point to be measured at time zero. Add 200 microliters of cells to each micro tube and incubate the cultures with shaking at 30 degrees Celsius.Then at each of the experimental time points, add 30 microliters of freshly prepared cyclo heide to one of the micro tubes. While the micro tubes are incubating, add 400 microliters of PBS to one corresponding fax tube for each micro tube. After the incubation, briefly vortex the micro tubes and then sonicate the cells and add water bath for one minute as just demonstrated.Then add 100 microliters of cell culture from each micro tube to its corresponding fax tube at the flow of cytometer. Load the proper excitation and detection settings and test the non expressing and fully expressing samples to verify that they fall in the detector sensitivity range. Finally, measure 10, 000 cells for each sample in these images, yeast cells bearing the expression reporter quadruple venous protein under the control of the TL one promoter and attached to the bottom of a well slide were stimulated by direct edition of stress medium.As just demonstrated, the cells were monitored for about two hours at 10 minute intervals, and images were acquired both before and after edition of the stimulus. Note, the fluorescent expression of the report during the activated cells. To extract the quantitative information contained in these images, a complex image analysis process must be performed here.The result obtained with the yeast quant analysis platform are shown. Each red trace represents the temporal evolution of the average fluorescence intensity of a single cell. The blue line illustrates the median of more than 500 cells that were segmented and tracked throughout 20 frames of five time lapse movies acquired from five different fields of view from the same well the light blue area represent the 25th and 75th percentile of all the intensities measured at a given time point to study the dynamics of the expression of the same reporter by flow cytometry Cycloheximide was added to the yeast cell cultures at different time points at five to 10 minute intervals.The drug blocks the synthesis of new proteins, but the maturation of the fluorescent protein already expressed is not perturbed. Therefore, the protein expression visualized by the displacement of the histogram to the right can be quantified at the time of Cycl H edition circumventing the problems associated with fluorescent protein maturation. In this graph, the dynamics of the expression reporter as measured by microscopy or flow cytometry are compared while the fluorescent signal rises 30 to 40 minutes after edition of the stress medium as measured by the microscopy, the flow cytometry measurements clearly indicate that the proteins are already produced between 10 to 15 minutes after the stimulus demonstrating the slow maturation of the fluorescent proteins.Both techniques allow access to single cell information for these simple experiments, the variability between three biological replicates is small compared to the large variability observed in the single cell responses by microscopy or flow cytometry. Following these procedures, other methods like those response measurements can be performed in order to answer additional questions such as, which is the threshold for gene expression. After watching this video, you should have a good understanding of how to measure protein expression at single cell level with microscope of flow cytometry.

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Learn temporal quantification of MAPK induced expression in single yeast cells using live cell microscopy and flow cytometry. This method enables precise measurement of gene expression dynamics in Saccharomyces cerevisiae.

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

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