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A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis

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Chromatin

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2458))

Abstract

Single-particle tracking (SPT) makes it possible to directly observe single protein diffusion dynamics in living cells over time. Thus, SPT has emerged as a powerful method to quantify the dynamics of nuclear proteins such as transcription factors (TFs). Here, we provide a protocol for conducting and analyzing SPT experiments with a focus on fast tracking (“fastSPT”) of TFs in mammalian cells. First, we explore how to engineer and prepare cells for SPT experiments. Next, we examine how to optimize SPT experiments by imaging at low densities to minimize tracking errors and by using stroboscopic excitation to minimize motion-blur. Next, we discuss how to convert raw SPT data into single-particle trajectories. Finally, we illustrate how to analyze these trajectories using the kinetic modeling package Spot-On. We discuss how to use Spot-On to fit histograms of displacements and extract useful information such as the fraction of TFs that are bound and freely diffusing, and their associated diffusion coefficients.

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Acknowledgments

We thank Domenic Narducci, Miles Huseyin, ** Yang, Hugo Brandão, Viraat Goel, Sarah Nemsick, Shdema Filler-Hayut, Michele Gabriele, Jyothi Mahadevan, Meagan Esbin, Maxime Woringer, and Thomas Graham for insightful comments on the manuscript. We would like to acknowledge Davide Mazza, whose 2012 paper introduced the kinetic modeling framework that was ultimately implemented in Spot-On in a modified form, Maxime Woringer who codeveloped Spot-On and led the development of the web-interface and the Python version and who has been maintaining the web-interface, the Tjian-Darzacq lab for discussions during the development of Spot-On and for hosting the web-interface, and Luke Lavis for the development and sharing of Janelia Fluor dyes. We thank Domenic Narducci for the code to simulate the concept of motion-blurring in Fig. 5. This work was supported by the National Institutes of Health under grant numbers R00GM130896, DP2GM140938, and UM1HG011536.

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Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Asmita Jha & Anders S. Hansen

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  1. Asmita Jha
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  2. Anders S. Hansen
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Correspondence to Anders S. Hansen .

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Editors and Affiliations

  1. Department of Pathology Dunedin School of Medicine, University of Otago, Dunedin, New Zealand

    Julia Horsfield

  2. Cardiology, Division Heart & Lungs, University Medical Center Utrecht, Utrecht, The Netherlands

    Judith Marsman

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Jha, A., Hansen, A.S. (2022). A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis. In: Horsfield, J., Marsman, J. (eds) Chromatin. Methods in Molecular Biology, vol 2458. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2140-0_9

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  • DOI: https://doi.org/10.1007/978-1-0716-2140-0_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2139-4

  • Online ISBN: 978-1-0716-2140-0

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