The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb3 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 Gb3 clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb3 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/Gb3 (65∶30∶5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb3 (40∶35∶20∶5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb3, STxB-Gb3 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.

The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga holotoxin, binds specifically to the glycosphingolipid Gb 3. In biological membranes, Gb 3 glycosphingolipids differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb 3 complex. To analyse the binding of STxB to different Gb 3 s, we chemically synthesized saturated, unsaturated, a-hydroxylated Gb 3 s and a combination thereof, all based on a C 24-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb 3 s were inserted into solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple mimetic of the outer leaflet of animal cell membranes. By fluorescenceand atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb 3 significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb 3. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb 3 s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb 3 s. Large interphase lipid redistribution is observed for a-hydroxylated Gb 3 doped membranes. Our results clearly demonstrate that the fatty acid of Gb 3 strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes.

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-β-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).

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Integrins are adhesion molecules that connect a cell to its environment and transduce signals bidirectionally across the membrane. Their different functional states correspond to distinct conformations. Using a biomembrane force probe, we observed real-time reversible switches between bent and extended conformations of single cell surface integrin aLb2 by measuring its nanometer-scale headpiece displacements, (un)bending frequencies and molecular stiffness changes. We determined stabilities of these conformations, their dynamic equilibrium, speeds and rates of conformational changes, and impacts of divalent cations and tensile forces. We quantified how initial conformations and their subsequent changes of aLb2 regulate the force-dependent kinetics of dissociation from intercellular adhesion molecule 1. Our findings provide new insights into how integrins function as nanomachines to precisely control cell adhesion and signaling.