Lipid Phase Separation and Protein-Ganglioside Clustering in Supported Bilayers Are Induced by Photorelease of Ceramide.

Photolysis of 6-bromo-7-hydroxycoumarinyl-caged ceramide was used to generate ceramide with spatial and temporal control in supported lipid bilayers prepared from mixtures of caged ceramide and phospholipids. The caged ceramide molecules are randomly distributed in fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, and upon photolysis with long wavelength UV light small ordered ceramide domains are formed that phase separate from the bulk fluid membrane. Irradiation of a spatially restricted area leads to the transient formation of ceramide-enriched gel phase domains that equilibrate via lipid diffusion with the surrounding unirradiated membrane. Photorelease of C16-ceramide in supported bilayers prepared from POPC, caged ceramide and the ganglioside GM1 (90:10:1 molar ratio) results in partitioning of a ganglioside-protein complex into the ceramide-enriched domains, modeling some aspects of ceramide’s behavior in cells. The photo-uncaging strategy used here for delivery of ceramide in bilayers provides a novel and useful alternative to the enzymatic generation of ceramide in sphingomyelin-containing membranes. The ability to control membrane phase separation behavior and the clustering of membrane-anchored proteins illustrates the potential of photo-uncaging for studying the compartmentalization of ceramide in cellular membranes.

在由笼锁神经酰胺和磷脂混合物制备的支撑脂质双层中，利用6 - 溴 - 7 - 羟基香豆素基笼锁神经酰胺的光解作用，在时空上可控地产生神经酰胺。笼锁神经酰胺分子随机分布在流体1 - 棕榈酰 - 2 - 油酰 - sn - 甘油 - 3 - 磷酸胆碱（POPC）双层中，用长波长紫外光光解时，会形成小的有序神经酰胺区域，这些区域与主体流体膜相分离。对空间受限区域进行照射会导致富含神经酰胺的凝胶相区域短暂形成，这些区域通过脂质扩散与周围未照射的膜达到平衡。在由POPC、笼锁神经酰胺和神经节苷脂GM1（摩尔比为90 : 10 : 1）制备的支撑双层中，C16 - 神经酰胺的光释放导致神经节苷脂 - 蛋白质复合物分配到富含神经酰胺的区域，模拟了神经酰胺在细胞中的某些行为。此处用于在双层中递送神经酰胺的光解笼锁策略为在含鞘磷脂的膜中酶促产生神经酰胺提供了一种新颖且有用的替代方法。控制膜相分离行为以及膜锚定蛋白质聚集的能力说明了光解笼锁在研究细胞膜中神经酰胺区室化方面的潜力。