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

eLife

Kabir Hassan Biswas¹  Dongmyung Oh²  Ronen Zaidel-Bar³  Jay T Groves⁴  Zhongwen Chen⁵ 

[1] College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar;Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, Galveston, United States;Mechanobiology Institute, National University of Singapore, Singapore, Singapore;Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel;Department of Chemistry, University of California, Berkeley, Berkeley, United States;Multiscale Research Institute of Complex Systems, Fudan University, Shanghai, China;Department of Chemistry, University of California, Berkeley, Berkeley, United States;

关键词: EphA2;    micropattern;    single molecule imaging;    receptor clustering;    supported membrane;    phase separation;    Human;   

DOI  :  10.7554/eLife.67379

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY