1. Physics of Living Systems
  2. Structural Biology and Molecular Biophysics

High-throughput single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking

  1. Yerim Lee
  2. Carey Phelps
  3. Tao Huang
  4. Barmak Mostofian
  5. Lei Wu
  6. Ying Zhang
  7. Kai Tao
  8. Young Hwan Chang
  9. Philip JS Stork
  10. Joe W Gray
  11. Daniel M Zuckerman  Is a corresponding author
  12. Xiaolin Nan  Is a corresponding author
  1. Oregon Health and Science University, United States
https://doi.org/10.7554/eLife.46393
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  1. Yerim Lee
  2. Carey Phelps
  3. Tao Huang
  4. Barmak Mostofian
  5. Lei Wu
  6. Ying Zhang
  7. Kai Tao
  8. Young Hwan Chang
  9. Philip JS Stork
  10. Joe W Gray
  11. Daniel M Zuckerman
  12. Xiaolin Nan
(2019)
High-throughput single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking
eLife 8:e46393.
https://doi.org/10.7554/eLife.46393
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Abstract

Membrane nanodomains have been implicated in Ras signaling, but what these domains are and how they interact with Ras remain obscure. Here, using single particle tracking with photoactivated localization microscopy (spt-PALM) and detailed trajectory analysis, we show that distinct membrane domains dictate KRasG12D (an active KRas mutant) diffusion and trafficking in U2OS cells. KRasG12D exhibits an immobile state in ~70 nm domains, each embedded in a larger domain (~200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRasG12D is continuously removed from the membrane via the immobile state and replenished to the fast state, reminiscent of Ras internalization and recycling. Importantly, both the diffusion and trafficking properties of KRasG12D remain invariant over a broad range of protein expression levels. Our results reveal how membrane organization dictates membrane diffusion and trafficking of Ras and offer new insight into the spatial regulation of Ras signaling.

Data availability

We have provided a complete set of model parameters derived from all raw single-particle tracking videos in the supplementary information.

Article and author information

Author details

  1. Yerim Lee

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Carey Phelps

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Tao Huang

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Barmak Mostofian

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0568-9866

  5. Lei Wu

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Ying Zhang

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Kai Tao

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.

  8. Young Hwan Chang

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Philip JS Stork

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. Joe W Gray

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9225-6756

  11. Daniel M Zuckerman

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    For correspondence
    zuckermd@ohsu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7662-2031

  12. Xiaolin Nan

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    For correspondence
    nan@ohsu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0597-0255

Funding

National Institutes of Health (U54 CA209988)

  • Young Hwan Chang
  • Joe W Gray
  • Xiaolin Nan

National Science Foundation (MCB1715823)

  • Daniel M Zuckerman

Damon Runyon Cancer Research Foundation

  • Xiaolin Nan

M J Murdock Charitable Trust

  • Joe W Gray
  • Xiaolin Nan

Prospect Creek Foundation

  • Joe W Gray
  • Xiaolin Nan

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

Copyright

© 2019, Lee 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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  1. Yerim Lee
  2. Carey Phelps
  3. Tao Huang
  4. Barmak Mostofian
  5. Lei Wu
  6. Ying Zhang
  7. Kai Tao
  8. Young Hwan Chang
  9. Philip JS Stork
  10. Joe W Gray
  11. Daniel M Zuckerman
  12. Xiaolin Nan
(2019)
High-throughput single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking
eLife 8:e46393.
https://doi.org/10.7554/eLife.46393

Share this article

https://doi.org/10.7554/eLife.46393

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Further reading

    1. Cancer Biology
    2. Physics of Living Systems

    Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior

    Debanjan Goswami, De Chen ... Thomas Turbyville
    Research Article Updated

    The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable region side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here, we characterize RAS in live human and mouse cells using single-molecule-tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein’s globular domain. To understand the altered mobility of an oncogenic KRAS4b, we used complementary experimental and molecular dynamics simulation approaches to reveal a detailed mechanism.