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/Gb(3) 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- (l(o)) and liquid-disordered (l(d)) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb(3) (40:35:20:5) membranes. The size of the l(o)-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-ß-cyclodextrin. Binding of Shiga toxin B-pentamers to the Gb(3)-doped membranes increased the l(o)-phase considerably and even induced l(o)-phase domains in non-phase separated bilayers composed of DOPC/sphingomyelin/cholesterol/Gb(3) (65:10:20:5).
Subject(s)
Lipid Bilayers/chemistry , Cholesterol/chemistry , Gold/chemistry , Microscopy, Fluorescence , Phase Transition , Phosphatidylcholines/chemistry , Porosity , Protein Binding , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Shiga Toxin 2/chemistry , Shiga Toxin 2/metabolism , Silicon/chemistry , Sphingomyelins/chemistry , Temperature , Unilamellar Liposomes/chemistry , beta-Cyclodextrins/chemistryABSTRACT
The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb(3) in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb(3) clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb(3) lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb(3) (65:30:5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb(3) (40:35:20:5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb(3), STxB-Gb(3) complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.
