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Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

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Chromosome Architecture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1431))

Abstract

The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA-binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types.

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Acknowledgments

Rodrigo Reyes-Lamothe, David J. Sherratt, and Achillefs N. Kapanidis helped with the original development of this protocol. Katarzyna Ginda and David J. Sherratt are thanked for their comments on the manuscript. Stephan Uphoff was funded by a Sir Henry Wellcome Postdoctoral Fellowship by the Wellcome Trust (101636/Z/13/Z) and a Junior Research Fellowship at St. John’s College, Oxford. Microscopy at Micron Oxford was supported by a Wellcome Trust Strategic Award (091911) and MRC grant (MR/K01577X/1).

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Authors and Affiliations

  1. Department of Biochemistry, University of Oxford, Parks Road, Oxford, OX1 3QU, UK

    Stephan Uphoff

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  1. Stephan Uphoff
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Correspondence to Stephan Uphoff .

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Editors and Affiliations

  1. Biological Physical Sciences Instit, University of York, York, United Kingdom

    Mark C. Leake

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Uphoff, S. (2016). Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial 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_16

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  • DOI: https://doi.org/10.1007/978-1-4939-3631-1_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3629-8

  • Online ISBN: 978-1-4939-3631-1

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