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

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Book cover Chromosome Architecture

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

Abstract

Single-molecule localization 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, 3D SMLM, and protein labeling, 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 analyzing 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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Acknowledgments

The manuscript was written by MS, IA, SES, VC, AP, DH, and SB. The figures shown were made by MS, IA, and VC. We would like to thank the Trinity College Stem-Cell Medicine Senior Postdoctoral Researcher Fund and the Wellcome Trust/MRC Cambridge Stem Cell Intitute for funding SB. We thank the Wellcome Trust 4-Year PhD Programme in Stem Cell Biology and Medicine for funding MS. SS is funded by a Joint Research Grant from the Isaac Newton Trust, Wellcome Trust ISSF, University of Cambridge, and the CSCI Wellcome Trust seed fund. Finally, we would like to thank Yi Lei Tan, Edward J.R. Taylor, Prof Ernest D. Laue, and Dr. Steven F. Lee for contributions to an earlier version of this book chapter. We would like to thank Prof Ernest D. Laue and Prof Sir David Klenerman for generous use of the microscopes on which these images were collected.

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Authors and Affiliations

  1. Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

    Maike Steindel, Igor Orsine de Almeida, Stanley Strawbridge, Valentyna Chernova & Srinjan Basu

  2. Group of Computational Biology and Applied Mathematics, Institute of Biology, Ecole Normale Supérieure, Paris, France

    David Holcman

  3. School of Physics and Astronomy and School of Food Science and Nutrition, University of Leeds, Leeds, UK

    Aleks Ponjavic

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  1. Maike Steindel
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Correspondence to Aleks Ponjavic or Srinjan Basu .

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

    Mark C. Leake

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Steindel, M. et al. (2022). Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localization Microscopy. 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_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2221-6_16

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