Sphingomyelin (SM), a major component of small domains (or lipid rafts) in mammalian cell membranes, forms a liquid-ordered phase in the presence of cholesterol (Cho). However, the nature of molecular interactions within the ordered SM/Cho phase remains elusive. We previously revealed that stearoyl-SM (SSM) and its enantiomer (ent-SSM) separately form nano-subdomains within the liquid-ordered phase involving homophilic SSM-SSM and ent-SSM-ent-SSM interactions. In this study, the details of the subdomain formation by SSMs at the nanometer range were examined using Förster resonance energy transfer (FRET) measurements in lipid bilayers containing SSM and ent-SSM, dioleoyl-phosphatidylcholine and Cho. Although microscopy detected a stereochemical effect on partition coefficient favoring stereochemically homophilic interactions in the liquid-ordered state, it showed no significant difference in large-scale liquid-ordered domain formation by the two stereoisomers. In contrast to the uniform domains seen microscopy, FRET analysis using fluorescent donor- and acceptor-labeled SSM showed distinct differences in SM and ent-SM colocalization within nanoscale distances. Donor- and acceptor-labeled SSM showed significantly higher FRET efficiency than did donor-labeled SSM and acceptor-labeled ent-SSM in lipid vesicles composed of “racemic” (1:1) mixtures of SSM/ent-SSM with dioleoylphosphatidylcholine and Cho. The difference in FRET efficiency indicated that SSM and ent-SSM assemble to form separate nano-subdomains. The average size of the subdomains decreased as temperature increased, and at physiological temperatures, the subdomains were found to have a single-digit nanometer radius. These results suggest that (even in the absence of ent-SM) SM-SM interactions play a crucial role in forming nano-subdomains within liquid-ordered domains and may be a key feature of lipid microdomains (or rafts) in biological membranes.

中文翻译：

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鞘磷脂（和 ent-sphingomyelin）在液体有序域内形成同质纳米级子域

鞘磷脂 (SM) 是哺乳动物细胞膜中小结构域（或脂筏）的主要成分，在胆固醇 (Cho) 存在下形成液体有序相。然而，有序 SM/Cho 相内分子相互作用的性质仍然难以捉摸。我们之前揭示了硬脂酰-SM (SSM) 及其对映异构体 (ent-SSM) 在涉及同质 SSM-SSM 和 ent-SSM-ent-SSM 相互作用的液体有序相内分别形成纳米亚域。在这项研究中，在含有 SSM 和 ent-SSM、二油酰-磷脂酰胆碱和 Cho 的脂质双层中，使用 Förster 共振能量转移 (FRET) 测量检查了 SSM 在纳米范围内形成子域的细节。尽管显微镜检测到分配系数的立体化学效应有利于液体有序状态下的立体化学同亲相互作用，但它显示两种立体异构体在大规模液体有序域形成方面没有显着差异。与显微镜观察到的均匀域相比，使用荧光供体和受体标记的 SSM 进行 FRET 分析显示出纳米级距离内 SM 和 ent-SM 共定位的明显差异。在由 SSM/ent-SSM 与二油酰磷脂酰胆碱和 Cho 的“外消旋”（1:1）混合物组成的脂质囊泡中，供体和受体标记的 SSM 显示出比供体标记的 SSM 和受体标记的 ent-SSM 显着更高的 FRET 效率。FRET 效率的差异表明 SSM 和 ent-SSM 组装形成单独的纳米子域。子域的平均大小随着温度的升高而减小，并且在生理温度下，发现子域具有个位数的纳米半径。这些结果表明（即使在没有 ent-SM 的情况下）SM-SM 相互作用在形成液体有序域内的纳米亚域中起着至关重要的作用，并且可能是生物膜中脂质微域（或筏）的关键特征。