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

Zhang, R; Fruhwirth, GO; Coban, O; Barrett, JE; Burgoyne, T; Lee, SH; Simonson, PD; ... Selvin, PR; + view all (2016) Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-Resolution Microscopy. ACS Nano , 11 (1) pp. 249-257. 10.1021/acsnano.6b05356. Green open access

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Abstract

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 intra-cluster distances (d). From this 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>50nm 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<50nm) 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.

Type: Article
Title: Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-Resolution Microscopy
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1021/acsnano.6b05356
Publisher version: http://dx.doi.org/10.1021/acsnano.6b05356
Language: English
Additional information: This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Nano, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see: http://dx.doi.org/10.1021/acsnano.6b05356.
Keywords: Bayesian modeling; cell-to-cell heterogeneity; EGFR; MAPK; super-resolution microscopy
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Oncology
URI: https://discovery.ucl.ac.uk/id/eprint/1528213
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