Molecular dissection of organization and dynamics of lipid domains in biomembranes

(1)脂质探针的特性Lysenin是一种能与鞘磷脂(SM)特异性结合的致孔毒素。毒素与膜的结合伴随着蛋白质的齐聚，导致孔的形成。尽管先前的一项研究表明，SM/胆固醇脂质体在抑制赖氨酸诱导的溶血方面比SM脂质体有效10,000倍，但胆固醇的作用并不是很清楚。我们的结果表明，胆固醇和SM/Lysenin的比例都是通过改变蛋白质齐聚状态来控制Lysenin单体的数量，从而影响溶血。杜拉霉素是一种与肉桂霉素密切相关的19个氨基酸的四环抗生素，已知它能结合磷脂酰乙醇胺(PE)。硬霉素的脂类专一性尚未确定。我们的研究表明，在中国仓鼠卵巢(CHO)细胞中，当细胞达到融合时，细胞胆固醇对内吞作用的调节作用增强。我们发现，几种荧光脂质的内吞途径是以细胞融合依赖的方式改变的。细胞胆固醇在细胞融合依赖性内吞作用中的关键作用被提出。我们的结果还表明，胆固醇通过Rab11控制一组膜脂的内吞途径。(3)内体脂域对细胞胆固醇稳态的调节利用D-PDMP和神经节苷脂GM1，表明富含BMP的晚期内体脂域的结构变化在细胞胆固醇积累中起关键作用。我们还使用了抗骨形态发生蛋白的单抗来检测骨形态发生蛋白结构域在巨噬细胞胆固醇稳态中的作用。

(1) Characterization of lipid probesLysenin is a pore-forming toxin that specifically binds sphingomyelin (SM). The binding of the toxin to the membrane is accompanied by the oligomerization of the protein, leading to pore formation. Although a previous study showed that SM/cholesterol liposomes were 10,000 times more effective than SM liposomes in inhibiting lysenin-induced hemolysis, the role of cholesterol is not precisely clarified. Our results indicate that both cholesterol and the SM/lysenin ratio control the amount of lysenin monomer via altering the state of protein oligomerization, thus affecting hemolysis.Duramycin is a 19 amino acid tetracyclic lantibiotic closely related to cinnamycin, which is known to bind phosphatidyl-ethanolamine (PE). The lipid specificity of duramycin was not established. Our study indicates that both duramycin and cinnamycin exclusively bind to ethanolamine phospholipids in curvature-dependent manner.(2) Regulation of endocytosis by cholesterolCellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We showed that the endocytic pathways of several fluorescent lipids were altered in cell confluency-dependent manner. The crucial role of cellular cholesterol in cell-confluency dependent endocytosis was suggested. Our results also suggest that cholesterol controls endocytic routes of a subset of membrane lipids through rab11.(3) Regulation of cellular cholesterol homeostasis by endosome lipid domainsUsing D-PDMP and ganglioside GM1, it is shown that the structure alteration of bis (monoacylglycero) phosphate (BMP)-rich late endosome lipid domain plays a crucial role in cellular cholesterol accumulation. We also employed anti-BMP monoclonal antibody to examine the role of BMP domain in cholesterol homeostasis in macrophages.