betadex and 1-2-oleoylphosphatidylcholine

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes

Topics: beta-Cyclodextrins; Cryoelectron Microscopy; Detergents; Escherichia coli; Escherichia coli Proteins; Hydrophobic and Hydrophilic Interactions; Ion Channels; Lipid Bilayers; Mechanotransduction, Cellular; Membranes, Artificial; Models, Molecular; Mutation; Nanostructures; Phosphatidylcholines; Protein Conformation

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.

Topics: Antineoplastic Agents, Phytogenic; beta-Cyclodextrins; Biological Transport; Cholestenones; Cholesterol; Digitonin; Dimyristoylphosphatidylcholine; Erythrocyte Membrane; Fluoresceins; Glycyrrhizic Acid; Hemolysis; Humans; Membrane Lipids; Oleanolic Acid; Ornithogalum; Phosphatidylcholines; Saponins; Unilamellar Liposomes

Membrane nanodomains are dynamic liquid entities surrounded by another type of dynamic liquid. Diffusion can take place inside, around and in and out of the domains, and membrane components therefore continuously shift between domains and their surroundings. In the plasma membrane, there is the further complexity of links between membrane lipids and proteins both to the extracellular matrix and to intracellular proteins such as actin filaments. In addition, new membrane components are continuously delivered and old ones removed. On top of this, cells move. Taking all of this into account imposes great methodological challenges, and in the present chapter we discuss some methods that are currently used for membrane nanodomain studies, what information they can provide and their weaknesses.

Topics: 2-Naphthylamine; Actin Cytoskeleton; beta-Cyclodextrins; Cell Membrane; Cholesterol; Cytoskeleton; Fluorescent Dyes; Humans; Jurkat Cells; Laurates; Membrane Proteins; Octoxynol; Phosphatidylcholines; Sphingomyelins; Unilamellar Liposomes

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).

Topics: beta-Cyclodextrins; Cholesterol; Gold; Lipid Bilayers; Microscopy, Fluorescence; Phase Transition; Phosphatidylcholines; Porosity; Protein Binding; Receptors, Cell Surface; Shiga Toxin 2; Silicon; Sphingomyelins; Temperature; Unilamellar Liposomes

We report a simple method to obtain stable asymmetric giant unilamellar vesicles (GUVs). Fluorescence correlation spectroscopy was used to quantitatively characterize vesicle properties. After brain sphingomyelin (bSM) was exchanged into dioleoylphosphatidylcholine (DOPC) GUVs, lateral diffusion in the bSM-containing outer leaflet decreased, whereas that in the DOPC-containing inner leaflet was largely unchanged, confirming asymmetry and a lack of coupling between the physical states of the inner and outer leaflets. In contrast, after bSM was exchanged into brain phosphatidylcholine vesicles, lateral diffusion decreased in both leaflets. Thus, asymmetric GUVs should be useful for investigating the molecular mechanisms behind interleaflet coupling.

Topics: beta-Cyclodextrins; Brain; Diffusion; Lipids; Phosphatidylcholines; Spectrometry, Fluorescence; Sphingomyelins; Unilamellar Liposomes

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-beta-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-beta-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.

Topics: Animals; beta-Cyclodextrins; Cattle; Cholesterol; Erythrocytes; Humans; Kinetics; Liposomes; Membrane Lipids; Membranes, Artificial; Perforin; Phosphatidylcholines; Surface Plasmon Resonance