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

  • Chiara Paviolo
  • James W. M. Chon
  • Andrew H. A. Clayton
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1112)

Abstract

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.

Keywords

Gold nanoparticles Epidermal growth factor receptor Phosphorylation Higher-order oligomers Y845 Y1173 MAPK Image correlation spectroscopy 

Notes

Acknowledgements

AHAC and JWMC gratefully acknowledge the Australian Research Council for funding this research (Grant Number: DP130101475).

Disclosure

The author reports no conflicts of interest in this work.

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Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Centre for Micro-Photonics, Faculty of Science, Engineering and TechnologySwinburne University of TechnologyHawthornAustralia

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