Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling.
Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling.
复制标题
通过冷冻断裂复制标记揭示跨膜磷脂分布。
作者：
K. Fujimoto;M. Umeda;T. Fujimoto
我们建议使用冷冻断裂的膜分裂来对原生质和外质膜小叶（一半）进行差异磷脂分析。未固定的细胞或组织被快速冷冻、冻裂和铂碳 (Pt/C) 阴影。然后用 2.5% 十二烷基硫酸钠 (SDS) 处理 Pt/C 复制品，以溶解未破裂的膜并释放细胞质或内容物。虽然去污剂溶解未破裂的膜，但它不会从裂开的膜中提取脂质，因为它们的非极性域通过其 Pt/C 复制品来稳定。清洗后，Pt/C 复制品以及附着的原生质和外质膜半部经过处理，用抗体对磷脂进行免疫细胞化学标记，然后进行电子显微镜观察。在这里，我们介绍了 SDS 消化的冷冻断裂复制标记 (SDS-FRL) 技术在各种细胞和细胞内膜中主要膜磷脂磷脂酰胆碱 (PC) 跨膜分布的应用。免疫金标记显示 PC 专门位于质膜的外质膜半部以及各种细胞器的细胞内膜上，例如细胞器。感光细胞的细胞核、线粒体、内质网、分泌颗粒和盘膜。这一总体方案的一个例外是形成神经元髓鞘的质膜和经 Ca(2+) 处理的红细胞膜。在这些细胞膜中，在外膜和内膜的每一半上都看到了大致等量的 PC 免疫金颗粒，这意味着 PC 的跨膜分布是对称的。初步筛选表明，SDS-FRL 技术允许对膜脂的跨膜分布进行原位分析，同时开辟了标记膜（例如通常无法进行细胞化学标记的细胞内膜）的可能性，而不会造成与膜分离程序相关的潜在扭曲。
We propose the use of membrane splitting by freeze-fracture for differential phospholipid analysis of protoplasmic and exoplasmic membrane leaflets (halves). Unfixed cells or tissues are quick-frozen, freeze-fractured, and platinum-carbon (Pt/C) shadowed. The Pt/C replicas are then treated with 2.5% sodium dodecyl sulfate (SDS) to solubilize unfractured membranes and to release cytoplasm or contents. While the detergent dissolves unfractured membranes, it would not extract lipids from split membranes, as their apolar domains are stabilized by their Pt/C replicas. After washing, the Pt/C replicas, along with attached protoplasmic and exoplasmic membrane halves, are processed for immunocytochemical labeling of phospholipids with antibody, followed by electron microscopic observation. Here, we present the application of the SDS-digested freeze-fracture replica labeling (SDS-FRL) technique to the transmembrane distribution of a major membrane phospholipid, phosphatidylcholine (PC), in various cell and intracellular membranes. Immunogold labeling revealed that PC is exclusively localized on the exoplasmic membrane halves of the plasma membranes, and the intracellular membranes of various organelles, e.g. nuclei, mitochondria, endoplasmic reticulum, secretory granules, and disc membranes of photoreceptor cells. One exception to this general scheme was the plasma membrane forming the myelin sheath of neurons and the Ca(2+)-treated erythrocyte membranes. In these cell membranes, roughly equal amounts of immunogold particles for PC were seen on each outer and inner membrane half, implying a symmetrical transmembrane distribution of PC. Initial screening suggests that the SDS-FRL technique allows in situ analysis of the transmembrane distribution of membrane lipids, and at the same time opens up the possibility of labeling membranes such as intracellular membranes not normally accessible to cytochemical labels without the distortion potentially associated with membrane isolation procedures.
低渗溶血过程中人红细胞膜磷脂的跨膜重新分布。
DOI：
10.1016/0005-2736(92)90176-m
发表时间：
1992
期刊：
Biochimica et biophysica acta
影响因子：
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作者：
Schrier,SL;Zachowski,A;Hervé,P;Kader,JC;Devaux,PF
通讯作者：
Devaux,PF