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Measuring the Co-Localization and Dynamics of Mobile Proteins in Live Cells Undergoing Signaling Responses

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The Immune Synapse

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2654))

Abstract

Single molecule imaging in live cells enables the study of protein interactions and dynamics as they participate in signaling processes. When combined with fluorophores that stochastically transition between fluorescent and reversible dark states, as in super-resolution localization imaging, labeled molecules can be visualized in single cells over time. This improvement in sampling enables the study of extended cellular responses at the resolution of single molecule localization. This chapter provides optimized experimental and analytical methods used to quantify protein interactions and dynamics within the membranes of adhered live cells. Importantly, the use of pair-correlation functions resolved in both space and time allows researchers to probe interactions between proteins on biologically relevant distance and timescales, even though fluorescence localization methods typically require long times to assemble well-sampled reconstructed images. We describe an application of this approach to measure protein interactions in B cell receptor signaling and include sample analysis code for post-processing of imaging data. These methods are quantitative, sensitive, and broadly applicable to a range of signaling systems.

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Acknowledgments

We thank Andrea Stoddard and Kathleen Wisser for contributing reagents and help with sample preparation. Jennifer Flanagan-Natoli developed protocols to functionalize coverglass for cell adhesion. Matthew Stone helped develop data analysis, sample preparation, and imaging methods. This work was funded by NIH (GM110052, GM134949, CA226962) and NSF (MCB1552439) grants to SLV. SAS was supported by an American Cancer Society postdoctoral fellowship grant (PF1800401CCE).

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Authors and Affiliations

  1. Program in Biophysics, University of Michigan, Ann Arbor, MI, USA

    Sarah A. Shelby & Sarah L. Veatch

  2. Program in Applied Physics, University of Michigan, Ann Arbor, MI, USA

    Thomas R. Shaw

Authors
  1. Sarah A. Shelby
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  2. Thomas R. Shaw
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  3. Sarah L. Veatch
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Correspondence to Sarah L. Veatch .

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Editors and Affiliations

  1. Department of Life Sciences, University of Siena, Siena, Italy

    Cosima T. Baldari

  2. Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK

    Michael L. Dustin

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Shelby, S.A., Shaw, T.R., Veatch, S.L. (2023). Measuring the Co-Localization and Dynamics of Mobile Proteins in Live Cells Undergoing Signaling Responses. In: Baldari, C.T., Dustin, M.L. (eds) The Immune Synapse. Methods in Molecular Biology, vol 2654. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3135-5_1

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  • DOI: https://doi.org/10.1007/978-1-0716-3135-5_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3134-8

  • Online ISBN: 978-1-0716-3135-5

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