It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-beta-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15 mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37 degrees C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23 degrees C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37 degrees C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.
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[Show abstract][Hide abstract] ABSTRACT: Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. Hence, it is a good starting point for studying the influence of lipid-protein interactions on membrane trafficking is to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. By measuring equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-β-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. Sterol partitioning was measured in DLPC, DMPC and DPPC bilayers with and without WALP19, WALP23 or WALP27 peptides. The results showed that the affinity of the sterol for the bilayers was affected by hydrophobic matching. An increasing positive hydrophobic mismatch led to stronger sterol binding to the bilayers (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. In addition, peptide insertion into the phospholipid bilayers was observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid-protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This could be of importance in membrane trafficking.
Full-text · Article · Dec 2012 · Biochimica et Biophysica Acta
"For fluorescence measurements, multilamellar vesicles were prepared by probe sonication (sonicated for 2 min with 20% duty cycle and 15 W power output) using a Branson probe sonifier (W-450, Branson Ultrasonics, Danbury, CT, USA). Unilamellar vesicles used for the CTL partitioning assay were prepared by extrusion (200 nm pore diameter) as described previously  "
[Show abstract][Hide abstract] ABSTRACT: Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1(Δ4t)), and N-linked acyl chains (often 16:0, 18:0 or 24:1(Δ15c)). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.
"Recently, we modified the cholesterol/cyclodextrin partitioning method of Niu and Litman , to use CTL instead of radioactive cholesterol (at 2 mol%). Furthermore, in our partitioning assay, there is no need to separate acceptor vesicles from cyclodextrin  , which makes measurements easier to perform. With this method we have shown that CTL partitions more favorably to PSM/POPC (20:80) containing bilayers as compared to POPC alone. "
[Show abstract][Hide abstract] ABSTRACT: We have prepared palmitoyl sphingomyelin (PSM) analogs in which either the 2-NH was methylated to NMe, the 3-OH was methylated to OMe, or both were methylated simultaneously. The aim of the study was to determine how such modifications in the membrane interfacial region of the molecules affected interlipid interactions in bilayer membranes. Measuring DPH anisotropy in vesicle membranes prepared from the SM analogs, we observed that methylation decreased gel-phase stability and increased fluid phase disorder, when compared to PSM. Methylation of the 2-NH had the largest effect on gel-phase instability (T(m) was lowered by ~7°C). Atomistic molecular dynamics simulations showed that fluid phase bilayers with methylated SM analogs were more expanded but thinner compared to PSM bilayers. It was further revealed that 3-OH methylation dramatically attenuated hydrogen bonding also via the amide nitrogen, whereas 2-NH methylation did not similarly affect hydrogen bonding via the 3-OH. The interactions of sterols with the methylated SM analogs were markedly affected. 3-OH methylation almost completely eliminated the capacity of the SM analog to form sterol-enriched ordered domains, whereas the 2-NH methylated SM analog formed sterol-enriched domains but these were less thermostable (and thus less ordered) than the domains formed by PSM. Cholestatrienol affinity to bilayers containing methylated SM analogs was also markedly reduced as compared to its affinity for bilayers containing PSM. Molecular dynamics simulations revealed further that cholesterol's bilayer location was deeper in PSM bilayers as compared to the location in bilayers made from methylated SM analogs. This study shows that the interfacial properties of SMs are very important for interlipid interactions and the formation of laterally ordered domains in complex bilayers.