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

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