Abstract
Single-molecule narrow-field 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 subcellular compartments. We describe how to obtain single-molecule microscopy data but also how to analyze 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 behavior at the whole cell and population level.
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Acknowledgments
We thank Sviatlana Shashkova and Stefan Hohmann (University of Gothenburg, Sweden) for donation of yeast cell strains and assistance with yeast cell culturing. M.C.L. was assisted by a Royal Society URF and research funds from the Biological Physical Sciences Institute (BPSI) of the University of York, UK.
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Wollman, A.J.M., Leake, M.C. (2016). Single-Molecule Narrow-Field Microscopy of Protein–DNA Binding Dynamics in Glucose Signal Transduction of Live Yeast Cells. In: Leake, M. (eds) Chromosome Architecture. Methods in Molecular Biology, vol 1431. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3631-1_2
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DOI: https://doi.org/10.1007/978-1-4939-3631-1_2
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