Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
J Struct Biol. 2011 Jul;175(1):21-30. doi: 10.1016/j.jsb.2011.03.021. Epub 2011 Apr 5.
Lipid microdomains, also called lipid rafts, consisting of sphingolipids and cholesterol, play important roles in membrane trafficking and in signaling. Despite years of study of the composition, size, half-life and dynamic organization of these domains, many open questions remain about their precise characteristics. To address some of these issues, we have developed a new experimental approach involving the use of specific monoclonal antibodies as recognition tools. One such antibody was raised against a homogeneous, mixed, ordered monolayer phase comprised of 60:40 mol% cholesterol:C16-ceramide, and has been used previously to demonstrate the existence of C16-ceramide/cholesterol domains in the membranes of cultured cells. We now use a combination of quantitative fluorescence microscopy, immuno-transmission electron microscopy and immuno-scanning cryo-electron microscopy, optimized for the study of intracellular lipid antigens. In a variety of cultured cells, C16-ceramide/cholesterol structural domains were found at high levels in late endosomes and in the trans-Golgi network, but were not found at statistically significant levels in early endosomes, lysosomes or the endoplasmic reticulum. We discuss the relevance of these results to understanding the role of lipid lateral organization in biological membranes.
脂质微区，也称为脂质筏，由鞘脂和胆固醇组成，在膜运输和信号转导中发挥重要作用。尽管多年来一直研究这些区域的组成、大小、半衰期和动态组织，但关于它们的确切特征仍存在许多悬而未决的问题。为了解决其中的一些问题，我们开发了一种新的实验方法，涉及使用特定的单克隆抗体作为识别工具。其中一种抗体是针对由 60:40mol%胆固醇：C16-神经酰胺组成的均匀、混合、有序单层相而产生的，以前曾用于证明培养细胞的膜中存在 C16-神经酰胺/胆固醇域。我们现在使用定量荧光显微镜、免疫透射电子显微镜和免疫扫描冷冻电子显微镜的组合，针对细胞内脂质抗原进行了优化。在各种培养的细胞中，C16-神经酰胺/胆固醇结构域在晚期内体和反式高尔基体网络中高水平存在，但在早期内体、溶酶体或内质网中不存在统计学上显著水平。我们讨论了这些结果对理解脂质侧向组织在生物膜中的作用的相关性。
