Dynamische reorganisatie van het plasmamembraan bij stimulatie van het individuele molecuul in vivo
01 / 2001 - 01 / 2005
Dynamic organization of proteins and lipids in the plasma membrane of cells plays a vital role in numerous biological processes. Many proteins involved in initial steps of cell signaling like G-protein coupled receptors (GPCRs), G-proteins, and RAS are attached or recruited to the membrane via lipid molecules. Plasma membrane localization of the signaling proteins is crucial for their function. There is strong evidence that the plasma membrane is not homogeneous, but distinctly structured into domains containing specific sets of proteins and lipids, some of them known as detergent-resistant domains, caveolae, and rafts. It has been proposed that those domains function as dynamic signaling depots, where GPCRs, G-proteins, RAS, etc., are clustered. The distribution of receptors into such compartments will control their accessibility to ligands and signaling molecules like G-proteins, hence, probably be an important regulatory mechanism. Although rafts have been inferred from functional and kinetic studies of intact cells, most evidence of their existence is based on differential extraction of cells with detergent. The relationship between membrane extracts and the in vivo composition and structure of lipid rafts is uncertain and controversial, also because of the lack of sensitive techniques to investigate the interaction between individual molecules. Aim of the current project is to use the newly developed single-molecule microscopy to follow in real-time the dynamic reorganization of the plasma membrane on single-molecule stimulation. We will concentrate on the first event of signal transduction, the recognition of a ligand (fluorescent labeled cAMP) by a receptor (G-protein coupled cAMP receptor fused to GFP) in vivo. Correlation with simultaneous observation of GFP fused to CAAX- and PH-domains (genetical membrane localization signals) will allow us to obtain information on the dynamics of receptor distribution, ligand-receptor interactions and membrane organization in a living cell during chemotaxis and persistent stimulation.