High-throughput microfluidics to control and measure signaling dynamics in single yeast cells

Abstract

Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure, and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy, and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. Using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, whilst collecting data for thousands of single cells. Compared to other protocols, the present protocol is relatively easy to adopt and higher-throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms.

A transcription factor, MrMsn2, in the dimorphic fungus Metarhizium rileyi is essential for dimorphism transition, aggravated pigmentation, conidiation and microsclerotia formation

“…3). In response to ROS stress, Msn2/4 accumulated in the nucleus, where they promoted transcriptional activation of stress‐responsive genes (Hansen et al ., 2015; Yi and Huh, 2015). However, deletion of MrMsn2 increased production of ROS (data not shown) and upregulated expression of antioxidant genes to protect against ROS.…”

“…They are similar to ScMsn2/4 of Saccharomyces cerevisiae (Schmitt and Mcentee, 1996) and have been characterized in Aspergillus parasiticus , A. flavus , Beauveria bassiana , Magnaporthe oryzae , Metarhizium robertsii and Verticillium dahliae (Chang et al ., 2011; Liu et al ., 2013; Zhang et al ., 2014; Tian et al ., 2017). Under abiotic and biotic stresses, Msn2/4 is phosphorylated for translocation from the cytoplasm to the nucleus, where it drives the transcription of stress‐induced genes (Hansen et al ., 2015; Yi and Huh, 2015; Li et al ., 2017). The underlying mechanism of the regulation of Msn2/4 activity by protein kinase A (PKA), the rapamycin signalling pathway, the Snf1 protein kinase pathway and the high‐osmolarity glycerol (HOG) pathway have been identified (Liu et al ., 2013; Zhang et al ., 2014; Li et al ., 2017).…”

Reconstructing dynamic molecular states from single-cell time series

“…Cells were then immediately imaged using the exact same exposure conditions as described in [6]. Images were then analysed and fluorescence quantified as previously described [6,51]. After quantifying the fluorescence intensity per cell for each of the five genes, we then fitted a simple line to the data and found that each YFP molecule contributed about 100.8 arb.…”

“…After quantifying the fluorescence intensity per cell for each of the five genes, we then fitted a simple line to the data and found that each YFP molecule contributed about 100.8 arb. units fluorescence per cell under our excitation settings [51]. We therefore divided the total fluorescence per cell from the previous dataset [6] to obtain the total number of YFP molecules per cell.…”

“…Interface 13: 20160533 pKT-vector; available from AddGene as no. 64685) followed by a HIS-marker by transforming a polymerase chain reaction-generated mCitrineV163A-HIS construct into the original haploid S288C Saccharomyces cerevisiae strain used by Ghaemmaghami et al [51] (EY0986, MAT a, ATCC201388, his31, leu20, met150, ura30, S288C) and selecting on SD-HIS plates. To minimize experimental variability, we picked and measured four independent clones for each of the five genes.…”