Mapping Epidermal Growth Factor Receptor (EGFR) Dimerization by Imaging Fluorescence Cross-Correlation Spectroscopy

Abstract

The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, plays a role in a number of important cellular processes such as migration, differentiation and proliferation. Although EGFR has been studied intensively for many years, some aspects of its activation and function have not been satisfactorily explained. For example, the dimerization of EGFR in the absence of its ligand remains controversial. Predominantly monomeric to predominantly dimeric and possibly higher oligomeric states have been reported in literature. In this study we have used a novel imaging modality called dual-colour imaging total internal reflection fluorescence cross-correlation spectroscopy (DC-ITIR-FCCS) to map EGFR dimerization in the membranes of CHO-K1 cells. The cells were expressing EGFR analogues tagged by eGFP and mRFP, respectively, and for each image pixel we measured the cross-correlation between fluorescence intensity fluctuations in the green and the red channel. High cross-correlation means co-diffusion of molecules tagged with eGFP and mRFP, which indicates presence of dimers or possibly higher oligomers. Our results show levels of cross-correlation consistent with considerable fractions of EGFR in the form of dimers. There exist, however, considerable differences between individual cells, some displaying negligible while others almost complete dimerization. The imaging FCCS modality allows us to evaluate not only the differences between individual cells but also the distribution of EGFR dimers within the membrane of each individual cell, showing for example increased dimerization close to the bounderies of the cells. Furthermore, the method allows us to map the differences in EGFR dimerization and its spatial distribution following EGFR activation or extraction of cholesterol from the membrane.