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

Cell-membrane sheets suitable for in-vitro functional fluorescence studies have been prepared by direct detachment from cell membranes Using poly-L-lysine-coated glass slides. The resulting transferred planar membranes conserve the composition 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-associated proteins, opening the possibility of labeling them and modulating their properties with membrane impermeable compounds. Second, the cytosolic autofluorescence of the cells is absent, allowing ultrasensitive measurements to be performed down to the single-molecule level. Third, the complexity of cellular processes occurring at the plasma membrane can be reduced, allowing the sequential investigation of selected events from complex biochemical networks. These advantages are illustrated by ligand-binding studies on the alpha 1b-adrenergic receptor. Our results indicate that supported membrane sheets might find a broad application as ail ideal in-vitro system for the elucidation of complex signaling pathways.