Epidermal Growth Factor Receptor (EGFR) Membrane Organization and Dynamics Investigated by Sw-Fccs and Imaging Fcs

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in various cellular processes including cell differentiation, proliferation, and migration. Despite many years of intensive research its mode of activation is still not fully understood, partly because its dimerization and its membrane localization are still under discussion. Here we use Single Wavelength excitation Fluorescence Cross-Correlation Spectroscopy (SW-FCCS) and Imaging Fluorescence Correlation Spectroscopy (Imaging FCS), to investigate receptor dimerization and receptor localization in membranes.The fraction of EGFR preformed dimers, i.e. dimerization in the absence of ligands, has been determined under various conditions with very different results, ranging from exclusive monomeric states to distributions between monomers dimers and possibly higher oligomers. The reason for these discrepancies is unclear. We therefore measure the dimerization on different cell lines (CHO-K1, Cos7, HEK293) at different membrane locations (basal and apical membrane) and at room and physiological temperature to determine whether dimerization is sensitive to these different conditions. We observe a cell line specific sensitivity of dimerization to temperature and membrane localization that can change the detected dimer fraction by more than a factor of 2, hinting at an explanation why different experiments provide different answers to the question of preformed dimers. Furthermore, we use Imaging FCS to investigate the localization of the EGFR within the membrane and its interaction with the cytoskeleton. Our study shows that the EGFR is interacting with cholesterol dependent and independent domains and its motion is influenced by the cytoskeleton. Overall these measurements provide a complex picture of EGFR organization and dynamics, which might play essential roles in receptor function and signaling.