High-throughput single-cell analysis for the proteomic dynamics study of the yeast osmotic stress response

Abstract

Motorized fluorescence microscopy combined with high-throughput microfluidic chips is a powerful method to obtain information about different biological processes in cell biology studies. Generally, to observe different strains under different environments, high-throughput microfluidic chips require complex preparatory work. In this study, we designed a novel and easily operated high-throughput microfluidic system to observe 96 different GFP-tagged yeast strains in one switchable culture condition or 24 different GFP-tagged yeast strains in four parallel switchable culture conditions. A multi-pipette is the only additional equipment required for high-throughput patterning of cells in the chip. Only eight connections are needed to control 96 conditions. Using these devices, the proteomic dynamics of the yeast stress response pathway were carefully studied based on single-cell data. A new method to characterize the proteomic dynamics using a single cell's data is proposed and compared to previous methods, and the new technique should be useful for studying underlying control networks. Our method provides an easy and systematic way to study signaling pathways at the single-cell level.

Figure 1. Microfluidic chip for high-throughput single-cell observation.

(a) Chip design. (b) Chip application method.

Figure 2. Data collection and image analysis platform.

(a) The signal pathway for yeast osmotic stress response. (b) Yeast strains were cultured in 96-well plates. (c) Cells were loaded into the chip using an 8-channel pipette. (d) Images were obtained by microscope. (e) Data statistical analysis.

Figure 3. Original fluorescence images at the single-cell level.

(a) The original fluorescence images of TPS2 and ALD4 (0.4 M KCl or 0.8 M KCl was added at 180 min). (b) TPS2 and ALD4 expression in osmotic stress response at the single-cell level.

Figure 4. The protein expression dynamics under osmotic stress (log base 2 of the normalized expression level).

(a) 0.2 M KCl, (b) 0.4 M KCl, (c) 0.8 M KCl. The stimuli were added at 30 min.

Figure 5. Single-cell analysis of the protein expression patterns.

(a) Single-cell analysis of HSP1; (b) Single-cell analysis of PGM2. The stimuli were added at 0 min.

Figure 6. Cluster analysis using the proteomic dynamic data from a single cell (log base 2 of the normalized expression level in 0.4 M KCl; the stimuli were added at 30 min).