The Dynamics and Localization of the Epidermal Growth Factor Receptor (EGFR) on Live Cell Plasma Membrane Studied by ITIR-FCS

Abstract

The Epidermal Growth Factor Receptor (EGFR), a prototypical receptor tyrosine kinase, plays an important role in cellular processes such as proliferation, migration and apoptosis. EGFR is known to be strongly regulated by its localization and the state of activation on the plasma membrane. However, the spatial distribution of EGFR on the plasma membrane has not been resolved in detail.Here, we use Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) to investigate EGFR behavior on live cells before and after stimulation. ITIR-FCS allows monitoring EGFR dynamics on cell membranes and in combination with the FCS diffusion laws provides information on the nanoscopic EGFR organization.Our results indicate that EGFR is partly trapped in cholesterol sensitive domains and also couples to the cytoskeleton. Stimulation by EGF activates EGFR in a dose dependent manner. At low-dose stimulation (10 ng/ml), the membrane diffusion and confinement of EGFR remain unchanged and EGFR internalization is cholesterol independent and EGFR recycles back to the membrane. At high-dose EGF treatment (100 ng/ml), EGFR endocytosis is cholesterol dependent and cholesterol-depletion impairs receptor internalization. In addition, cholesterol-depletion with concomitant EGF stimulation leads to stronger confinement of the receptor, independent of the EGF dose, as clusters accumulate on the membrane. Furthermore, the actin cytoskeleton promotes EGFR internalization after ligand stimulation. Disruption of the actin cytoskeleton inhibits EGFR endocytosis and induces clustering of EGFR on the cell membrane. This study demonstrates the capabilities of ITIR-FCS to follow receptor stimulation events on live cells. It demonstrates not only that EGFR activation is cholesterol and cytoskeleton dependent but also that the activation pathway is EGF dose dependent, thus providing new insights into receptor dynamics and organization on live cells.