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

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submitted on 2024-05-22, 08:27 and posted on 2024-05-22, 08:27 authored by Zhongwen Chen, Dongmyung Oh, Kabir Hassan Biswas, Ronen Zaidel-Bar, Jay T Groves

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 1 µm, 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.

Other Information

Published in: eLife
License: http://creativecommons.org/licenses/by/4.0/
See article on publisher's website: https://dx.doi.org/10.7554/elife.67379

Funding

Open Access funding provided by the Qatar National Library.

History

Language

  • English

Publisher

eLife Sciences Publications

Publication Year

  • 2021

License statement

This Item is licensed under the Creative Commons Attribution 4.0 International License.

Institution affiliated with

  • Hamad Bin Khalifa University
  • College of Health and Life Sciences - HBKU

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