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Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy

[Image: see text] Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locati...

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Autores principales: Zhang, Ruobing, Fruhwirth, Gilbert O., Coban, Oana, Barrett, James E., Burgoyne, Thomas, Lee, Sang Hak, Simonson, Paul Dennis, Baday, Murat, Kholodenko, Boris N., Futter, Clare E., Ng, Tony, Selvin, Paul R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2016
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5269639/
https://www.ncbi.nlm.nih.gov/pubmed/27768850
http://dx.doi.org/10.1021/acsnano.6b05356
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author Zhang, Ruobing
Fruhwirth, Gilbert O.
Coban, Oana
Barrett, James E.
Burgoyne, Thomas
Lee, Sang Hak
Simonson, Paul Dennis
Baday, Murat
Kholodenko, Boris N.
Futter, Clare E.
Ng, Tony
Selvin, Paul R.
author_facet Zhang, Ruobing
Fruhwirth, Gilbert O.
Coban, Oana
Barrett, James E.
Burgoyne, Thomas
Lee, Sang Hak
Simonson, Paul Dennis
Baday, Murat
Kholodenko, Boris N.
Futter, Clare E.
Ng, Tony
Selvin, Paul R.
author_sort Zhang, Ruobing
collection PubMed
description [Image: see text] Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intracluster distances (d). From these data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P < 0.001). For comparison, the prediction accuracy was only 61% (P = 0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used. Large clusters (N ≥ 3) with d > 50 nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d < 50 nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells.
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spelling pubmed-52696392017-01-30 Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy Zhang, Ruobing Fruhwirth, Gilbert O. Coban, Oana Barrett, James E. Burgoyne, Thomas Lee, Sang Hak Simonson, Paul Dennis Baday, Murat Kholodenko, Boris N. Futter, Clare E. Ng, Tony Selvin, Paul R. ACS Nano [Image: see text] Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intracluster distances (d). From these data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P < 0.001). For comparison, the prediction accuracy was only 61% (P = 0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used. Large clusters (N ≥ 3) with d > 50 nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d < 50 nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells. American Chemical Society 2016-10-21 2017-01-24 /pmc/articles/PMC5269639/ /pubmed/27768850 http://dx.doi.org/10.1021/acsnano.6b05356 Text en Copyright © 2016 American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) , which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
spellingShingle Zhang, Ruobing
Fruhwirth, Gilbert O.
Coban, Oana
Barrett, James E.
Burgoyne, Thomas
Lee, Sang Hak
Simonson, Paul Dennis
Baday, Murat
Kholodenko, Boris N.
Futter, Clare E.
Ng, Tony
Selvin, Paul R.
Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy
title Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy
title_full Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy
title_fullStr Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy
title_full_unstemmed Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy
title_short Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy
title_sort probing the heterogeneity of protein kinase activation in cells by super-resolution microscopy
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5269639/
https://www.ncbi.nlm.nih.gov/pubmed/27768850
http://dx.doi.org/10.1021/acsnano.6b05356
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