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Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localisation Microscopy

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Chromosome Architecture

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

Abstract

Single-molecule localisation microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal ‘snapshots’ of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination and protein labelling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analysing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.

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Acknowledgements

We would like to thank the Royal Society for the University Research Fellowship of Steven F. Lee (UF120277) and the Medical Research Council for the Research Fellowship of Srinjan Basu (MR/M010082/1). We would like to thank Brian Hendrich and David Klenerman for generous use of the cell culture facilities used to grow the mouse embryonic stem cells imaged here. The figures shown were made by Srinjan Basu with the help of Yi Lei Tan, Thomas A. Drury, Edward J.R. Taylor and Steven F. Lee. I would like to thank Ulrike Endesfelder, Kai Wohlfahrt, Melike Lakadamyali and David Lando for discussion and for critical reading of the manuscript.

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  1. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK

    Srinjan Basu, Yi Lei Tan, Edward J. R. Taylor, Ernest D. Laue & Steven F. Lee

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  1. Srinjan Basu
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  2. Yi Lei Tan
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  3. Edward J. R. Taylor
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Correspondence to Steven F. Lee .

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

    Mark C. Leake

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Basu, S., Tan, Y.L., Taylor, E.J.R., Laue, E.D., Lee, S.F. (2016). Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localisation Microscopy. 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_17

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  • DOI: https://doi.org/10.1007/978-1-4939-3631-1_17

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