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.

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.

Metrics

  • 1,968
    views
  • 288
    downloads
  • 23
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Further reading

    1. Physics of Living Systems
    Marcelo A Carignano, Martin Kroeger ... Igal Szleifer
    Research Article

    We propose the Self Returning Excluded Volume (SR-EV) model for the structure of chromatin based on stochastic rules and physical interactions. The SR-EV rules of return generate conformationally defined domains observed by single-cell imaging techniques. From nucleosome to chromosome scales, the model captures the overall chromatin organization as a corrugated system, with dense and dilute regions alternating in a manner that resembles the mixing of two disordered bi-continuous phases. This particular organizational topology is a consequence of the multiplicity of interactions and processes occurring in the nuclei, and mimicked by the proposed return rules. Single configuration properties and ensemble averages show a robust agreement between theoretical and experimental results including chromatin volume concentration, contact probability, packing domain identification and size characterization, and packing scaling behavior. Model and experimental results suggest that there is an inherent chromatin organization regardless of the cell character and resistant to an external forcing such as RAD21 degradation.

    1. Physics of Living Systems
    Yaojun Zhang, Andrew GT Pyo ... Ned S Wingreen
    Research Article

    A hallmark of biomolecular condensates formed via liquid-liquid phase separation is that they dynamically exchange material with their surroundings, and this process can be crucial to condensate function. Intuitively, the rate of exchange can be limited by the flux from the dilute phase or by the mixing speed in the dense phase. Surprisingly, a recent experiment suggests that exchange can also be limited by the dynamics at the droplet interface, implying the existence of an ‘interface resistance’. Here, we first derive an analytical expression for the timescale of condensate material exchange, which clearly conveys the physical factors controlling exchange dynamics. We then utilize sticker-spacer polymer models to show that interface resistance can arise when incident molecules transiently touch the interface without entering the dense phase, i.e., the molecules ‘bounce’ from the interface. Our work provides insight into condensate exchange dynamics, with implications for both natural and synthetic systems.