Ramirez Daniel M Carter, Ogilvie William W, Johnston Linda J
Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON K1A 0R6, Canada.
Biochim Biophys Acta. 2010 Mar;1798(3):558-68. doi: 10.1016/j.bbamem.2009.12.005. Epub 2009 Dec 21.
A series of cholesterol (Chol) probes with NBD and Dansyl fluorophores attached to the 3-hydroxyl position via carbamate linkers has been designed and synthesized and their ability to mimic the behavior of natural cholesterol in bilayer membranes has been examined. Fluorescence spectroscopy data indicate that the NBD-labeled lipids are located in the polar headgroup region of the bilayer with their position varying with the method of fluorophore attachment and the linker length. The partitioning of the Chol probes between liquid-ordered (L(o)) and liquid-disordered (L(o)) phases in supported bilayers prepared from ternary lipid mixtures of DOPC, Chol and either egg sphingomyelin or DPPC was examined by fluorescence microscopy. The carbamate-linked NBD-Chols show a stronger preference for partitioning into L(o) domains than does a structurally similar probe with an ester linkage, indicating the importance of careful optimization of probe and linker to provide the best Chol mimic. Comparison of the partitioning of NBD probes to literature data for native Chol indicates that the probes reproduce well the modest enrichment of Chol in L(o) domains as well as the ceramide-induced displacement of Chol. One NBD probe was used to follow the dynamic redistribution of Chol in phase separated membranes in response to in situ ceramide generation. This provides the first direct optical visualization of Chol redistribution during enzymatic ceramide generation and allows the assignment of new bilayer regions that exclude dye and have high lateral adhesion to ceramide-rich regions.
设计并合成了一系列胆固醇（Chol）探针，这些探针通过氨基甲酸酯连接子将NBD和丹磺酰荧光团连接到3-羟基位置，并研究了它们在双层膜中模拟天然胆固醇行为的能力。荧光光谱数据表明，NBD标记的脂质位于双层膜的极性头基区域，其位置随荧光团连接方法和连接子长度而变化。通过荧光显微镜检查了由DOPC、Chol和卵鞘磷脂或DPPC的三元脂质混合物制备的支持双层膜中Chol探针在液相有序（L(o)）和液相无序（L(d)）相之间的分配情况。与具有酯键的结构相似探针相比，氨基甲酸酯连接的NBD-Chols对分配到L(o)结构域表现出更强的偏好，这表明仔细优化探针和连接子对于提供最佳Chol模拟物很重要。将NBD探针的分配情况与天然Chol的文献数据进行比较表明，这些探针能够很好地重现Chol在L(o)结构域中的适度富集以及神经酰胺诱导的Chol位移。使用一种NBD探针跟踪相分离膜中Chol响应原位神经酰胺生成的动态重新分布情况。这首次直接通过光学手段可视化了酶促生成神经酰胺过程中Chol的重新分布，并确定了新的双层区域，这些区域排斥染料并与富含神经酰胺的区域具有高侧向粘附性。
