Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Summary

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.

Abstract

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.

Introduction

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 blots^1-3. 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 quantitati....

Protocol

1. Microscopy Measurements

1. Inoculate 5 ml of synthetic medium with yeast.
2. Grow cells at 30 °C overnight.
3. Measure the OD₆₀₀ of the overnight culture the next morning.
4. Dilute overnight culture in 5 ml synthetic medium to OD₆₀₀ 0.05.
5. Grow the diluted culture at 30 °C for at least 4 hr.
6. Prepare 3-fold concentrated (0.6 M) NaCl solution in synthetic medium.
7. Prepare a 1 mg/ml solution of Concanavalin A (ConA) in PBS.
8. Filtrate ~200 μl of ConA solution into the well slide.
9. Let stand for 30 min.
10. Remove ConA solution.
11. Add 150 μ....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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