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>
	<...

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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 border="1">
		<tbody>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Reagent</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></td>
			 <td></td>
			 <td></td>
			 <td>Material</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

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...

Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

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 ...

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Research

JoVE Journal

Biology

8.4K visualizações.  University of Lausanne.  Dois métodos complementares baseados em citometria de fluxo e microscopia são apresentadas, que permitem a quantificação, no nível de uma única célula, da dinâmica da expressão de genes induzidos pela activação de uma via de MAPK em levedura. 