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Creating and Modulating Microdomains in Pore-Spanning Membranes

Authors

  • Alexander Orth,

    1. Institut für Organische und Biomolekulare Chemie, Georg-August-Universität Göttingen, Tammannstr. 2, 37077 Göttingen (Germany)
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  • Dr. Ludger Johannes,

    1. Institut Curie, Centre de Recherche, CNRS UMR 144, Laboratoire Trafic, Signalisation et Ciblage Intracellulaires, 75248 Paris cedex 05 (France)
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  • Prof. Dr. Winfried Römer,

    1. Institut Curie, Centre de Recherche, CNRS UMR 144, Laboratoire Trafic, Signalisation et Ciblage Intracellulaires, 75248 Paris cedex 05 (France)
    2. Institut für Biologie II and Bioss—Centre for Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg (Germany)
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  • Prof. Dr. Claudia Steinem

    Corresponding author
    1. Institut für Organische und Biomolekulare Chemie, Georg-August-Universität Göttingen, Tammannstr. 2, 37077 Göttingen (Germany)
    • Institut für Organische und Biomolekulare Chemie, Georg-August-Universität Göttingen, Tammannstr. 2, 37077 Göttingen (Germany)
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Abstract

The architecture of the plasma membrane is not only determined by the lipid and protein composition, but is also influenced by its attachment to the underlying cytoskeleton. Herein, we show that microscopic phase separation of “raft-like” lipid mixtures in pore-spanning bilayers is strongly determined by the underlying highly ordered porous substrate. In detail, lipid membranes composed of DOPC/sphingomyelin/cholesterol/Gb3 were prepared on ordered pore arrays in silicon with pore diameters of 0.8, 1.2 and 2 μm, respectively, by spreading and fusion of giant unilamellar vesicles. The upper part of the silicon substrate was first coated with gold and then functionalized with a thiol-bearing cholesterol derivative rendering the surface hydrophobic, which is prerequisite for membrane formation. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liquid-ordered- (lo) and liquid-disordered (ld) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb3 (40:35:20:5) membranes. The size of the lo-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temperature, and the cholesterol content in the membrane, which was modulated by the addition of methyl-β-cyclodextrin. Binding of Shiga toxin B-pentamers to the Gb3-doped membranes increased the lo-phase considerably and even induced lo-phase domains in non-phase separated bilayers composed of DOPC/sphingomyelin/cholesterol/Gb3 (65:10:20:5).

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