Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility

  1. Zhongwen Chen
  2. Dongmyung Oh
  3. Kabir Hassan Biswas  Is a corresponding author
  4. Ronen Zaidel-Bar  Is a corresponding author
  5. Jay T Groves  Is a corresponding author
  1. Fudan University, China
  2. National University of Singapore, Singapore
  3. Hamad Bin Khalifa University, Qatar
  4. Tel Aviv University, Israel
  5. University of California, Berkeley, United States

Abstract

Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly one micron, and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances both receptor phosphorylation and downstream signaling activity. Single molecule imaging clearly resolves increased molecular binding dwell times at EphA2 clusters for both Grb2:SOS and NCK:N-WASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3, 4, and 5.

Article and author information

Author details

  1. Zhongwen Chen

    Fudan University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5218-0152
  2. Dongmyung Oh

    National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0817-5254
  3. Kabir Hassan Biswas

    Mechanobiology Institute, Hamad Bin Khalifa University, Doha, Qatar
    For correspondence
    kbiswas@hbku.edu.qa
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9194-4127
  4. Ronen Zaidel-Bar

    Tel Aviv University, Tel Aviv, Israel
    For correspondence
    zaidelbar@tauex.tau.ac.il
    Competing interests
    The authors declare that no competing interests exist.
  5. Jay T Groves

    QB3, University of California, Berkeley, Berkeley, United States
    For correspondence
    JTGroves@lbl.gov
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3037-5220

Funding

National Cancer Institute (Physical Sciences in Oncology Network Project 1-U01CA202241)

  • Zhongwen Chen
  • Jay T Groves

national science foundation Singapore (CRP001-084)

  • Zhongwen Chen
  • Dongmyung Oh
  • Ronen Zaidel-Bar
  • Jay T Groves

Shanghai Municipal Science and Technology Major Project (ZJLab (2018SHZDZX01))

  • Zhongwen Chen

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

Reviewing Editor

  1. Amy S Gladfelter, University of North Carolina at Chapel Hill, United States

Version history

  1. Received: February 9, 2021
  2. Preprint posted: February 17, 2021 (view preprint)
  3. Accepted: August 19, 2021
  4. Accepted Manuscript published: August 20, 2021 (version 1)
  5. Version of Record published: August 27, 2021 (version 2)

Copyright

© 2021, Chen 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. Zhongwen Chen
  2. Dongmyung Oh
  3. Kabir Hassan Biswas
  4. Ronen Zaidel-Bar
  5. Jay T Groves
(2021)
Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
eLife 10:e67379.
https://doi.org/10.7554/eLife.67379

Share this article

https://doi.org/10.7554/eLife.67379

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