The Effect of Nanoparticles on the Cluster Size Distributions of Activated EGFR Measured with Photobleaching Image Correlation Spectroscopy

The epidermal growth factor receptor (EGFR) is an important cell surface receptor in normal physiology and disease. Recent work has shown that EGF-gold nanoparticle conjugates can influence cell behaviour, but the underlying mechanism at the receptor quaternary structural level remains poorly understood.In the present work, the cluster density and cluster size of activated (phosphorylated) EGFR clusters in HeLa cells were determined with photobleaching image correlation spectroscopy. EGFR activation was probed via immunofluorescence-detected phosphorylation of tyrosines (pY-mAb) located in the kinase domain of EGFR (Y845) and at the EGFR cytoplasmic tail (Y1173). Cell activation was probed via nuclear extracellular-regulated kinase (ERK) phosphorylation. The cluster size of activated EGFR was 1.3-2.4 pY-mAb/cluster in unstimulated HeLa cells. EGF or nanorod treatment led to an increase in EGFR oligomers containing multiple phosphotyrosines (>2 phosphotyrosines per EGFR oligomer, average cluster size range = 3-5 pY-mAb/cluster) which paralleled increases in nuclear p-ERK. In contrast, EGF-nanorods decreased the contribution from higher-order phospho-clusters and decreased nuclear p-ERK relative to the nanorod control. These studies provide direct evidence that targeted nanotechnology can manipulate receptor organization and lead to changes in receptor activation and subsequent signalling processes.