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

  • Srinjan Basu
  • Yi Lei Tan
  • Edward J. R. Taylor
  • Ernest D. Laue
  • Steven F. LeeEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Chromatin Super-resolution microscopy PALM STORM SPT Fluorescence imaging SPIM Mean squared displacement Jump distance Residence time Diffusion coefficient 

Notes

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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Srinjan Basu
    • 1
  • Yi Lei Tan
    • 1
  • Edward J. R. Taylor
    • 1
  • Ernest D. Laue
    • 1
  • Steven F. Lee
    • 1
    Email author
  1. 1.Department of BiochemistryUniversity of CambridgeCambridgeUK

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