Observing Signal Transduction Directly at the Single-Molecule Level in Live Eukaryotic Cells

Abstract

All cells dynamically sense their environment through signal transduction mechanisms, allowing them to respond to environmental changes at the genetic level. The glucose sensory mechanism in Sacchoromyces cerevisiae is a model system of signal transduction. It uses the multi-copy inhibitor of the GAL gene expression Mig1 protein to repress unwanted genes in the presence of elevated level of extracellular glucose. In wild-type cell strains, and in genetically and biochemically impaired signal transduction phenotypes, we have fluorescently labelled the Mig1 protein with the green fluorescent protein GFP via chromosomal integration at native levels of expression, in addition to the RNAP nuclear reporter protein Nrd1 with the red fluorescent protein mCherry. [1] Using millisecond dual-color fluorescence excitation with Slimfield microscopy, [2] we track the fate of single molecules of Mig1 in live yeast cells at millisecond timescales. We present data showing a consistent difference between confined, high stoichiometry assemblages of bound Mig1 in the nucleus and freely diffusing single Mig1 molecules across the entire cell as well as more complex behavior in Mig1 traversing the nuclear membrane. The mobility and stoichiometry distribution of Mig1 assemblages we observe is functionally dependent on the signal transduction pathway and the concentration of extracellular glucose, allowing us to directly observe gene regulatory events.1. Bendrioua, L. et al. Yeast AMP-activated Protein Kinase Monitors Glucose Concentration Changes and Absolute Glucose Levels. J. Biol. Chem. 289, 12863-75 (2014).2. Reyes-Lamothe, R., Sherratt, D. J. & Leake, M. C. Stoichiometry and architecture of active DNA replication machinery in Escherichia coli. Science 328, 498-501 (2010).