Dietrich C, Bagatolli L A, Volovyk Z N, Thompson N L, Levi M, Jacobson K, Gratton E
Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, 27599, USA.
Biophys J. 2001 Mar;80(3):1417-28. doi: 10.1016/S0006-3495(01)76114-0.
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 degrees 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.
筏假说的一个关键原则是，富含糖鞘脂和胆固醇的脂域的形成可以仅由特征性的脂-脂相互作用驱动，这表明筏应该在由适当脂质组成的模型膜中形成。事实上，在平面支持脂质层以及由以下物质形成的巨大单层囊泡（GUV）中，都发现具有筏样性质的域与流体脂质区域共存：1）磷脂-胆固醇-鞘磷脂的等摩尔混合物，或2）从富含鞘磷脂和胆固醇的刷状缘膜中提取的天然脂质。在平面支持脂质层中使用头部基团标记的荧光磷脂类似物，通过荧光显微镜在室温（24摄氏度）下观察到直径通常为几微米的域，而非筏混合物（PC-胆固醇）则显得均匀。筏域和非筏域都呈流体状，尽管筏域中的扩散较慢，且探针可以在两相之间交换。与筏假说一致，糖鞘脂（GSL）GM1在更有序的域中高度富集，并且对去污剂提取具有抗性，去污剂会破坏GSL耗尽的相。为了排除域结构是由脂质层支持物引起的假象的可能性，用合成和天然脂质混合物形成了GUV，其中掺入了探针LAURDAN。通过双光子荧光显微镜检查LAURDAN的发射光谱，这允许识别脂质酰基链排列具有高或低有序度的区域。在由筏脂质混合物或刷状缘膜脂质形成的GUV中，在两个单层中对齐的一系列更有序和较无序的域可以在冷却和加热时可逆地形成和破坏。总体而言，这项研究有力地支持了这样一种观点，即在生物膜中，特定的脂质可以横向聚集形成更有序、抗去污剂的脂筏，糖鞘脂会分配到其中。
