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Single-Molecule Narrow-Field Microscopy of Protein-DNA Binding Dynamics in Glucose Signal Transduction of Live Yeast Cells

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Chromosome Architecture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2476))

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 levels.

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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. MCL was assisted by a Royal Society URF and research funds from the Biological Physical Sciences Institute (BPSI) of the University of York, UK.

Author information

Authors and Affiliations

  1. Newcastle University Biosciences Institute, Newcastle University, Newcastle, UK

    Adam J. M. Wollman

  2. Departments of Physics and Biology, University of York, York, UK

    Mark C. Leake

Authors
  1. Adam J. M. Wollman
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  2. Mark C. Leake
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Corresponding author

Correspondence to Adam J. M. Wollman .

Editor information

Editors and Affiliations

  1. Departments of Physics and Biology, University of York, York, UK

    Mark C. Leake

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Wollman, A.J.M., Leake, M.C. (2022). Single-Molecule Narrow-Field Microscopy of Protein-DNA Binding Dynamics in Glucose Signal Transduction of Live Yeast Cells. In: Leake, M.C. (eds) Chromosome Architecture. Methods in Molecular Biology, vol 2476. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2221-6_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2221-6_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2220-9

  • Online ISBN: 978-1-0716-2221-6

  • eBook Packages: Springer Protocols

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