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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 syste...

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Autores principales: Chen, Zhongwen, Oh, Dongmyung, Biswas, Kabir Hassan, Zaidel-Bar, Ronen, Groves, Jay T
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8397371/
https://www.ncbi.nlm.nih.gov/pubmed/34414885
http://dx.doi.org/10.7554/eLife.67379
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author Chen, Zhongwen
Oh, Dongmyung
Biswas, Kabir Hassan
Zaidel-Bar, Ronen
Groves, Jay T
author_facet Chen, Zhongwen
Oh, Dongmyung
Biswas, Kabir Hassan
Zaidel-Bar, Ronen
Groves, Jay T
author_sort Chen, Zhongwen
collection PubMed
description 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.
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spelling pubmed-83973712021-08-30 Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility Chen, Zhongwen Oh, Dongmyung Biswas, Kabir Hassan Zaidel-Bar, Ronen Groves, Jay T eLife Physics of Living Systems 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. eLife Sciences Publications, Ltd 2021-08-20 /pmc/articles/PMC8397371/ /pubmed/34414885 http://dx.doi.org/10.7554/eLife.67379 Text en © 2021, Chen et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Physics of Living Systems
Chen, Zhongwen
Oh, Dongmyung
Biswas, Kabir Hassan
Zaidel-Bar, Ronen
Groves, Jay T
Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
title Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
title_full Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
title_fullStr Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
title_full_unstemmed Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
title_short Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
title_sort probing the effect of clustering on epha2 receptor signaling efficiency by subcellular control of ligand-receptor mobility
topic Physics of Living Systems
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8397371/
https://www.ncbi.nlm.nih.gov/pubmed/34414885
http://dx.doi.org/10.7554/eLife.67379
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