Lipid rafts reconstituted in model membranes
Lipid rafts reconstituted in model membranes
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DOI：
10.1016/s0006-3495(01)76114-0
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发表时间：
2001-03-01
影响因子：
3.4
通讯作者：
Gratton, E
中科院分区：
文献类型：
--
作者：
Dietrich, C;Bagatolli, LA;Gratton, E
筏假说的一个关键原则是，鞘糖脂和胆固醇丰富的脂质结构域的形成可以单独由特征性的脂质-脂质相互作用驱动，这表明筏应该在由适当的脂质组成的模型膜中形成。事实上，发现具有筏状性质的结构域与流体脂质区域共存于平面支撑的脂质层和由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提取的天然脂质形成的巨大单层囊泡（GUV）中。在室温（24 ℃）下通过荧光显微镜观察到直径通常为几微米的区域，而非筏状混合物（IPC-胆固醇）看起来是均匀的。筏状和非筏状结构域都是流体状的，尽管在筏状结构域中扩散较慢，并且探针可以在两相之间交换。与筏假说一致，GM 1，鞘糖脂（GSL），高度富集在更有序的结构域和耐洗涤剂提取，这破坏了GSL耗尽相。为了排除结构域结构是由脂质层支持物引起的伪影的可能性，从合成和天然脂质混合物形成GUV，其中掺入探针LAURDAN。通过双光子荧光显微镜检查LAURDAN的发射光谱，可以识别脂酰基链排列顺序较高或较低的区域。在由筏脂质混合物或刷状缘膜脂质形成的GUV中，在两个单层中登记的更有序和更不有序的结构域的阵列可以可逆地形成并在冷却和加热时被破坏。总体而言，在生物膜中，选定的脂质可以横向聚集，形成更有序的，耐洗涤剂的脂筏，其中鞘糖脂分区的概念得到了本研究的有力支持。
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 degreesC) whereas non-raft mixtures IPC-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.