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

Ruobing Zhang, Gilbert O. Fruhwirth, Oana Coban, James E. Barrett, Thomas Burgoyne, Sang Hak Lee, Paul Dennis Simonson, Murat Baday, Boris N. Kholodenko, Clare E. Futter, Tony Ng, Paul R. Selvin

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)249-257
Number of pages9
JournalACS Nano
Volume11
Issue number1
DOIs
StatePublished - Jan 24 2017

Keywords

  • Bayesian modeling
  • EGFR
  • MAPK
  • cell-to-cell heterogeneity
  • super-resolution microscopy

ASJC Scopus subject areas

  • Materials Science(all)
  • Engineering(all)
  • Physics and Astronomy(all)

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  • Cite this

    Zhang, R., Fruhwirth, G. O., Coban, O., Barrett, J. E., Burgoyne, T., Lee, S. H., Simonson, P. D., Baday, M., Kholodenko, B. N., Futter, C. E., Ng, T., & Selvin, P. R. (2017). Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy. ACS Nano, 11(1), 249-257. https://doi.org/10.1021/acsnano.6b05356