Robust model-based analysis of single-particle tracking experiments with Spot-On

Abstract
Data availability
Article and author information
Metrics

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.

Data availability

The following data sets were generated

Article and author information

Author details

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.
Maxime Woringer

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-2581-9808
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.
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.
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.

"This ORCID iD identifies the author of this article:" 0000-0003-0539-8217
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.

"This ORCID iD identifies the author of this article:" 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.

Copyright

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.