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.

Age-dependent decline in stress response capacity revealed by proteins dynamics analysis

“…The protocol to load this chip is similar to the protocol described in our previous report work 21 . Cells of different strains are loaded into strain channels on the chip through the outlets using a pipette.…”

Robotic Cell Printing for Constructing Living Yeast Cell Microarrays in Microfluidic Chips

“…To create a living GFP-tagged yeast array, we combined mechanical patterning by constructing an array of microwells with cell printing (robotic cell patterning), which allows the controlled placement of the GFP-tagged clones in the selected wells. The cells were trapped in the wells by sticking them to the glass bottom of the microwells using the lectin Con A. Microfluidic chips where a GFP-tagged yeast clone collection was patterned as an array into microchambers have been previously developed [10,37]. Also, a microfluidic perfusion system where a robotic printed yeast array on agar and sandwiched with a track-etched membrane has been described [38].…”

“…As demonstrated here, dynamic movements from one location to another can also be followed as the cell proceeds through the cell cycle. Dynamic movements of proteins have also been described in yeast cells that respond to environmental stresses such as dithiothreitol (DTT) stress [9], hydrogen peroxide stress [9], osmotic stress by potassium chloride [37], and nitrogen starvation [9] or chemical perturbations by rapamycin [11], hydroxyurea [11], or methyl methane sulfonate [10].…”

Metabolic Cost of Rapid Adaptation of Single Yeast Cells

“…However, consistent with previous results 12 , the adaptation process observed here appears to differ from known S. cerevisiae stress responses, including the response to amino-acid starvation 23 , in a number of ways. Known stress responses are rapid and transient (with changes in gene expression typically peaking after <2 hours) 21,24 , and all cells respond broadly similarly 24 . In contrast, the characteristic timescale of adaptation of single cells that that we observe is much longer (~10 3 hours, see Fig.…”