Epidermal Growth Factor Receptor Localization in Cell Membranes Investigated by Imaging FCS

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays essential roles in cell growth and proliferation. Despite many years of research there are still open questions about its mechanism of function. We have addressed earlier the question of EGFR dimerization as well as phosphorylation in live cells. Here we address in particular its distribution and dynamics on the cell membrane. The simultaneous elucidation of membrane organization and dynamics requires measurements with good spatiotemporal resolution. We recently introduced Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) with time resolution down to 0.3 ms and diffraction-limited resolution to study whole membrane areas with single molecule sensitivity. ITIR-FCS can test the FCS diffusion laws and determine whether diffusion is free, takes place within a meshwork or is hindered by traps. We demonstrate the feasibility of this approach with various structured artificial bilayers. Advancing to live cell measurements, we compare the diffusive behavior of DiI (C12 and C18), a membrane marker for the fluid phase, GPI-GFP, a putative microdomain marker, transferring receptor-mCherry, a receptor interacting with the cytoskeleton, and the epidermal growth factor receptor-mRFP (EGFR-mRFP). We monitor the dynamics of the different molecules under conditions of cholesterol depletion (mbCD), cytoskeleton depolymerization (Latrunculin A), and EGF stimulation. EGFR clearly shows changes upon stimulation, implying stronger domain localization, with little sensitivity to depolymerization of the cytoskeleton and moderate sensitivity to cholesterol depletion.