Single molecule narrowfield microscopy of protein-DNA binding dynamics in glucose signal transduction of live yeast 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.
💡 Analysis
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.
📄 Content
Single molecule narrowfield microscopy of protein-DNA binding dynamics in glucose signal transduction of live yeast cells Adam J.M. Wollman1,2 and Mark C. Leake1
1 Biological Physical Sciences Institute (BPSI) University of York York YO10 5DD United Kingdom
2 Corresponding author e–mail: adam.wollman@york.ac.uk Tel: +44 (0)1904 322697
Running head: Single-molecule narrowfield.
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.
Key words: Single-molecule biophysics, signal-transduction, yeast
- Introduction
Bulk biochemical methods can only measure mean ensemble properties while single molecule techniques allow the heterogeneity in molecular biology to be explored which often leads to a new understanding of the biological system involved.(1) The use of fluorescent protein fusions to act as reporters can provide significant insight into a wide range of biological processes and molecular machines, for enabling insight into stoichiometry and architecture as well as details of molecular mobility inside living, functional cells with their native physiological context intact.(2–7) Single-molecule narrowfield microscopy, and its similar counterpart Slimfield microscopy, is a versatile tool to investigate a diverse range of protein dynamics in live cells which can be used in conjunction with fluorescent protein fusion strains to generate enormous insight into biological processes at the single-molecule level. In bacteria, it has been used to investigate the components of the replisome(8) and the structural maintenance of chromosomes.(9)
In narrowfield microscopy, the normal fluorescence excitation field is reduced to encompass only a single cell. This produces a Gaussian excitation field (∼30 μm2) with 100–1000 times the laser excitation intensity of standard epifluorescence microscopy. This intense illumination causes fluorophores to emit many more photons, generating much greater signal intensity relative to normal camera-imaging noise and, hence, facilitates millisecond time- scale imaging of single fluorescently-labelled proteins. The millisecond time scale is fast enough to keep up with the diffusional motion present in the cytoplasm of cells and can also sample the fast molecular transitions that occur, particularly during signal transduction. Single fluorescent proteins or complexes of proteins can be considered point sources of light and so appear as spatially extended spots in a fluorescence image due to diffraction by the microscope optics.(10) Narrowfield microscopy data consists of a time-series of images of spots which require a significant amount of in silico analysis. Spots must be identified by software, the intensity of these spots quantified to calculate their stoichiometry and their position tracked over time to produce a trajectory.
We have applied narrowfield microscopy to glucose signal transduction in budding yeast, Saccharomyces cerevisiae. All cells dynamically sense their environment through signal transduction mechanisms. The majority of these mechanisms rely on gene regulation through cascades of protein-protein interactions which transmit signals from sensory elements to responsive elements within each cell. The Mig1 protein is an essential transcription factor in this mechanism in yeast. Mig1 is a Cys2 -His2 zinc finger DNA binding protein(11) which binds several glucose-repressed promoters.(12–15) In the presence of extracellular glucose it is poorly phosphorylated and predominantly located in the nucleus(16, 17) where it recruits a repression complex to the DNA.(18) If extracellular glucose concentrations levels are depleted, Mig1 is phosphorylated by the sucrose non- fermenting protein (Snf1),(19–21) resulting in a redistribution of mean localization of Mig1 into the cytoplasm.(16, 22, 23) Thus, Mig1 concentration levels in the cell nucleus and cytoplasm serve as a readout of glucose signal transduction in budding yeast.(24) Mig1 has been labelled with the green fluorescent protein, GFP, and in the same strain, a ribosome component, Nrd1, almost completely localised to the nucleus, has been labelled with the mCherry fluorescen
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