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Unformatted text preview: Lipid Rafts Reconstituted in Model Membranes C. Dietrich,* L. A. Bagatolli, ² Z. N. Volovyk, ‡ N. L. Thompson, ‡ M. Levi, § K. Jacobson,* ,¶ and E. Gratton ² *Department of Cell Biology and Anatomy, ‡ Department of Chemistry, and ¶ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, ² Laboratory for Fluorescence Dynamics, Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and § Department of Medicine, University of Texas Southwestern Medical Center at Dallas and Veterans Administration Medical Center, Dallas, Texas 75216 USA ABSTRACT One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic 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 supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid- cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24°C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers 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....
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This note was uploaded on 01/12/2012 for the course BBA 2005 taught by Professor Staff during the Fall '11 term at University of Florida.
- Fall '11
- The Land