Supported cell-membrane sheets for functional fluorescence imaging of membrane proteins

Cell-membrane sheets suitable for in-vitro functional fluorescence studies have been prepd. by direct detachment from cell membranes using poly-L-lysine-coated glass slides. The resulting transferred planar membranes conserve the compn. as well as most properties of the original plasma membrane; in particular, both membrane leaflets remain fluid, allowing the investigation of diffusion properties of different cellular membrane components. Measurements on membrane sheets offer several advantages as compared to those on living cells. First, access to the intracellular leaflet is obtained, in particular to the intracellular part of membrane proteins and to cytoplasmic membrane-assocd. proteins, opening the possibility of labeling them and modulating their properties with membrane impermeable compds. Second, the cytosolic autofluorescence of the cells is absent, allowing ultrasensitive measurements to be performed down to the single-mol. level. Third, the complexity of cellular processes occurring at the plasma membrane can be reduced, allowing the sequential investigation of selected events from complex biochem. networks. These advantages are illustrated by ligand-binding studies on the a1b-adrenergic receptor. Our results indicate that supported membrane sheets might find a broad application as an ideal in-vitro system for the elucidation of complex signaling pathways. [on SciFinder (R)]