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Supported Cell-Membrane Sheets for Functional Fluorescence Imaging of Membrane Proteins

Authors

  • J.-B. Perez,

    1. Laboratory of Physical Chemistry of Polymers and Membranes, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
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  • K. L. Martinez,

    1. Laboratory of Physical Chemistry of Polymers and Membranes, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
    2. Present address: Bio-Nano Laboratory, Nano-Science Center, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
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  • J.-M. Segura,

    1. Laboratory of Physical Chemistry of Polymers and Membranes, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
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  • H. Vogel

    1. Laboratory of Physical Chemistry of Polymers and Membranes, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
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  • We thank the following colleagues providing plasmids for the particular receptors: Dr. Bruno Meyer for GFP-NK1R, Dr. Erwin Ilegems for 5HT3-CFP, and Prof. Susanna Cotecchia and Dr. Laura Stanasila for α1b-AR. This research was supported by Fonds National Suisse de la Recherche Scientifique (Grant 4047-057572).

Abstract

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 α1b-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.

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