Single-Molecule Transcription Factor Dynamics in Saccharomyces Cerevisiæ Glucose Sensing

Abstract

Metabolic processes underlie all forms of life. An organism's ability to utilise chemical energy to stay alive and eventually reproduce is a central feature of life, regardless of organism length scale. To achieve this, an organism must be able to adapt to varying surrounding environmental conditions. Cells respond to external stimuli by releasing kinase cascades along often intricate signalling pathways which regulate cellular function. These chemical signals eventually bring about some cell level response.Brewer's yeast, Saccharomyces cerevisiae is an extremely well characterised eukaryote model organism and many of its signal pathways have been studied extensively. The bulk behaviour of glucose sensing in yeast has recently been well characterised(1) but the dynamics and interaction of individual molecules in the pathway has not. We use several mutant yeast strains with fluorescent protein tags attached to the transcription factors Mig1 and Msn2 and the AMP kinase Snf1.We have developed a novel high speed fluorescence microscope system capable of imaging and tracking diffusing single or small clusters of fluorescent protein molecules. We have combined this with a bespoke microfluidic channel system allowing us to precisely change the extracellular glucose content and study the dynamics of signal transduction along the glucose sensing pathway in yeast. Our novel deconvolution method(2) let us count the total protein copy number by compartment and accurately follow a cascade interaction with transcription factors.1. Bendrioua L, Smedh M, Almquist J, Cvijovic M, Jirstrand M, Goksor M, et al. Yeast AMP-activated protein kinase monitors glucose concentration changes and absolute glucose levels. J Biol Chem. 2014;289(18):12863-75.2. Wollman AJM, Leake MC. Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time. Faraday Discuss. 2015 Jan;184:401-24.