Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingol ipid- and cholesterol-rich lipid domains can be driven solely by characteri stic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar su pported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1 ) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natura l lipids extracted from brush border membranes that are rich in sphingomyel in and cholesterol, Employing headgroup-labeled fluorescent phospholipid an alogs in planar supported lipid layers, domains typically several microns i n diameter were observed by fluorescence microscopy at room temperature (24 degreesC) whereas non-raft mixtures IPC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slo wer in raft domains, and the probe could exchange between the two phases. C onsistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was hig hly enriched in the more ordered domains and resistant to detergent extract ion, which disrupted the GSL-depleted phase. To exclude the possibility tha t the domain structure was an artifact caused by the lipid layer support, G UVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was exa mined by two-photon fluorescence microscopy, which allowed identification o f regions with high or low order of lipid acyl chain alignment. In GUVs for med from the raft lipid mixture or from brush border membrane lipids an arr ay of more ordered and less ordered domains that were in register in both m onolayers could reversibly be formed and disrupted upon cooling and heating . Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.