Measurement of molecular clustering in two-dimensional systems by fluorescence fluctuation spectroscopy: Application to EGF receptors on living cells

The overall aim of the work is to investigate the clustering of Epidermal Growth Factor (EGF) receptors on the surfaces of live cells under physiological conditions. This involves the extension and validation of fluorescence-intensity distribution analysis to a two-dimensional membrane system. The method is validated using measurements of fluorescent molecules of different brightness on model membrane bilayers. Monte Carl simulation were employed and data generated by simulation were used to characterize the dependence of the shape of the χ 2 surface on various parameters. Measurements in the absence of the physiological ligand, EGF, show that the clustering of EGF receptor on the cell membrane depends on the membrane cholesterol levels. Chemical cross linking of receptors was employed as an independent method to assess the clustering of EGF receptors and confirmed the results of brightness analysis. Measurements in the presence of EGF will show how binding of this ligand to the receptor influences the equilibrium between monomer and higher order clusters at cell surface.