title: Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy.
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 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.
authors: Ruobing
authors: R
authors: Zhang
authors: R. Dimbleby Department of Cancer Research, Randall Division of Cell and Molecular Biophysics, Division of Cancer Studies, King's College London , Guy's Campus New Hunt's House, London SE1 1UL, U.K.|Department of Imaging Chemistry and Biology, Division of Imaging Sciences and Biomedical Engineering, St. Thomas' Hospital, King's College London , London SE1 7EH, U.K.
authors: Gilbert O
authors: GO
authors: Fruhwirth
authors: R. Dimbleby Department of Cancer Research, Randall Division of Cell and Molecular Biophysics, Division of Cancer Studies, King's College London , Guy's Campus New Hunt's House, London SE1 1UL, U.K.
authors: Oana
authors: O
authors: Coban
authors: Department of Mathematics, King's College London , 25 Gordon Street, London WC2R 2LS, U.K.
authors: James E
authors: JE
authors: Barrett
authors: UCL Institute of Ophthalmology , 11-43 Bath Street, London EC1 V 9EL, U.K.
authors: Thomas
authors: T
authors: Burgoyne
authors: Sang Hak
authors: SH
authors: Lee
authors: Paul Dennis
authors: PD
authors: Simonson
authors: Murat
authors: M
authors: Baday
authors: Systems Biology Ireland, Conway Institute of Biomolecular & Biomedical Research, School of Medicine and Medical Science, University College Dublin , Belfield, Dublin 4, Ireland.
authors: Boris N
authors: BN
authors: Kholodenko
authors: UCL Institute of Ophthalmology , 11-43 Bath Street, London EC1 V 9EL, U.K.
authors: Clare E
authors: CE
authors: Futter
authors: R. Dimbleby Department of Cancer Research, Randall Division of Cell and Molecular Biophysics, Division of Cancer Studies, King's College London , Guy's Campus New Hunt's House, London SE1 1UL, U.K.|UCL Cancer Institute, Paul O'Gorman Building, University College London , London WC1E 6DD, U.K.|Breakthrough Breast Cancer Research Unit, Department of Research Oncology, Guy's Hospital King's College London School of Medicine , London SE1 9RT, U.K.
authors: Tony
authors: T
authors: Ng
authors: Paul R
authors: PR
authors: Selvin
mesh_terms: D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D016014:Linear Models; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D015335:Molecular Probes; D045663:Quantum Dots / Q000737:chemistry
chemical_list: D015335:Molecular Probes; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases
keywords: Bayesian modeling; EGFR; MAPK; cell-to-cell heterogeneity; super-resolution microscopy