Creating and Modulating Microdomains in Pore-Spanning Membranes

被引:25
作者
Orth, Alexander [1 ]
Johannes, Ludger [2 ]
Roemer, Winfried [2 ,3 ,4 ]
Steinem, Claudia [1 ]
机构
[1] Univ Gottingen, Inst Organ & Biomol Chem, D-37077 Gottingen, Germany
[2] CNRS UMR 144, Ctr Rech, Inst Curie, Lab Traf Signalisat & Ciblage Intracellulaires, F-75248 Paris 05, France
[3] Univ Freiburg, Inst Biol 2, D-79104 Freiburg, Germany
[4] Univ Freiburg, Bioss Ctr Biol Signalling Studies, D-79104 Freiburg, Germany
关键词
fluorescence microscopy; glycolipids; lipid domains; phase separation; porous silicon; MODEL MEMBRANES; PHASE-SEPARATION; LIPID-MEMBRANES; DYNAMICS; CHOLESTEROL; DIFFUSION; VESICLES; REORGANIZATION; PHOSPHOLIPIDS; MOBILITY;
D O I
10.1002/cphc.201100644
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
摘要
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 mu m, 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-beta-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).
引用
收藏
页码:108 / 114
页数:7
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