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

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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.

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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.

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Zhongwen Chen

Fudan University, Shanghai, China

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-5218-0152
Dongmyung Oh

National University of Singapore, Singapore, Singapore

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-0817-5254
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.

"This ORCID iD identifies the author of this article:" 0000-0001-9194-4127
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.
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.

"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

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.