The study of interactions of transmembrane receptors and intracellular signaling proteins in live cells by fluorescence correlation and cross-correlation spectroscopy

Abstract

Signal transduction between cells or inside cells is essential for organisms to coordinate their basic activities. To study interactions among signaling proteins is fundamental for discovery of novel signaling pathways and understanding of signaling mechanisms, which in turn may help in drug design. In this thesis we focused on applying biophysical fluorescence techniques, especially fluorescence correlation and cross-correlation spectroscopy, to investigate the interactions between members from epidermal growth factor receptor (EGFR/ErbB) family and the interactions between Cdc42 (for cell division cycle) and its effector proteins in live cells. Quantitation of protein-protein interactions in live cells is a challenge in present biological science. In this thesis we further developed single-wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS), established by Hwang and Wohland, as a new biophysical strategy to quantitatively study the interactions of the above two groups of signaling proteins in live cells. As a result, the dimerization percentages of ErbB receptors on cell membrane were determined, which may help in understanding the activation mechanism of ErbB receptors. Furthermore, the binding strengths between Cdc42 and its effectors were determined. In summary, this thesis reveals SW-FCCS as a novel tool for quantification of molecular interactions in live cells.