King's College London

Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identicalcells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remainspoorly understood. MAPK cascades integrate signals emanating from different EGFR spatiallocations, including the plasma membrane and endocytic compartment. We previouslyhypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level andactivity 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 defineEGFR 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 datareturning 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. Largeclusters (N≥3) with d>50nm were most predictive for pMAPK level in cells. Electronmicroscopy revealed that these large clusters were primarily localized to the limiting membraneof multivesicular bodies (MVB). Many tighter packed dimers/multimers (d<50nm) were foundon intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because ofthe physical separation. Our results suggest that cell-to-cell differences in N and d contain crucialinformation to predict EGFR-activated cellular pMAPK levels and explain pMAPKheterogeneity in isogenic cells.