Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
- Fudan University, China
- National University of Singapore, Singapore
- Hamad Bin Khalifa University, Qatar
- Tel Aviv University, Israel
- 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
Kabir Hassan Biswas
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.
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