Single molecule narrowfield microscopy of protein-DNA binding dynamics in glucose signal transduction of live yeast cells

TL;DR

This paper extends single-molecule narrowfield microscopy techniques to live yeast cells, enabling detailed analysis of protein-DNA interactions involved in glucose signal transduction and transcriptional regulation.

Contribution

It demonstrates how to adapt and analyze single-molecule microscopy data in larger eukaryotic cells, specifically yeast, for studying protein-DNA binding dynamics.

Findings

Successful adaptation of microscopy techniques to yeast cells

Ability to track and determine stoichiometry of molecular complexes

Insights into single-molecule regulation of transcription in live cells

Abstract

Single-molecule narrowfield microscopy is a versatile tool to investigate a diverse range of protein dynamics in live cells and has been extensively used in bacteria. Here, we describe how these methods can be extended to larger eukaryotic, yeast cells, which contain sub-cellular compartments. We describe how to obtain single-molecule microscopy data but also how to analyse these data to track and obtain the stoichiometry of molecular complexes diffusing in the cell. We chose glucose mediated signal transduction of live yeast cells as the system to demonstrate these single-molecule techniques as transcriptional regulation is fundamentally a single molecule problem - a single repressor protein binding a single binding site in the genome can dramatically alter behaviour at the whole cell and population level.