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