1. Biophysics and Structural Biology
  2. Computational and Systems Biology
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Robust model-based analysis of single-particle tracking experiments with Spot-On

  1. Anders S Hansen Is a corresponding author
  2. Maxime Woringer
  3. Jonathan B Grimm
  4. Luke D Lavis
  5. Robert Tjian Is a corresponding author
  6. Xavier Darzacq Is a corresponding author
  1. University of California, Berkeley, United States
  2. Howard Hughes Medical Institute, United States
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Cite as: eLife 2018;7:e33125 doi: 10.7554/eLife.33125

Abstract

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce 'Spot-On', an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.

Article and author information

Author details

  1. Anders S Hansen

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    anders.sejr.hansen@berkeley.edu
    Competing interests
    No competing interests declared.
  2. Maxime Woringer

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0003-2581-9808
  3. Jonathan B Grimm

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    Jonathan B Grimm, has filed patent applications (e.g. PCT/US2015/023953) whose value may be affected by this publication.
  4. Luke D Lavis

    Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    Luke D Lavis, has filed patent applications (e.g. PCT/US2015/023953) whose value may be affected by this publication.
  5. Robert Tjian

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    jmlim@berkeley.edu
    Competing interests
    Robert Tjian, One of the three founding funders of eLife and a member of eLife's Board of Directors.
    ORCID icon 0000-0003-0539-8217
  6. Xavier Darzacq

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    darzacq@berkeley.edu
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0003-2537-8395

Funding

National Institutes of Health (UO1-EB021236)

  • Xavier Darzacq

National Institutes of Health (U54-DK107980)

  • Xavier Darzacq

California Institute for Regenerative Medicine (LA1-08013)

  • Xavier Darzacq

Howard Hughes Medical Institute (3061)

  • Robert Tjian

Howard Hughes Medical Institute

  • Luke D Lavis

Siebel Stem Cell Institute

  • Anders S Hansen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. David Sherratt, Reviewing Editor, University of Oxford, United Kingdom

Publication history

  1. Received: October 27, 2017
  2. Accepted: January 3, 2018
  3. Accepted Manuscript published: January 4, 2018 (version 1)

Copyright

© 2018, Hansen et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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