Fujimoto K, Umeda M, Fujimoto T
Department of Anatomy, Faculty of Medicine, Kyoto University, Japan.
J Cell Sci. 1996 Oct;109 ( Pt 10):2453-60. doi: 10.1242/jcs.109.10.2453.
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.
我们建议使用冷冻断裂法进行膜分裂，以对原生质膜和外质膜小叶（半叶）进行差异性磷脂分析。未固定的细胞或组织经快速冷冻、冷冻断裂，然后进行铂-碳（Pt/C）镀膜。接着用2.5%的十二烷基硫酸钠（SDS）处理Pt/C复制品，以溶解未断裂的膜并释放细胞质或内含物。虽然去污剂能溶解未断裂的膜，但不会从分裂的膜中提取脂质，因为其非极性区域通过Pt/C复制品得以稳定。洗涤后，将Pt/C复制品连同附着的原生质膜和外质膜半叶一起进行处理，用抗体对磷脂进行免疫细胞化学标记，随后进行电子显微镜观察。在此，我们展示了SDS消化冷冻断裂复制品标记（SDS-FRL）技术在主要膜磷脂磷脂酰胆碱（PC）在各种细胞和细胞内膜中的跨膜分布研究中的应用。免疫金标记显示，PC仅定位在质膜的外质膜半叶以及各种细胞器的细胞内膜上，如细胞核、线粒体、内质网、分泌颗粒和光感受器细胞的盘状膜。这一普遍模式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，在每个外膜和内膜半叶上观察到大致等量的PC免疫金颗粒，这意味着PC呈对称的跨膜分布。初步筛选表明，SDS-FRL技术能够对膜脂的跨膜分布进行原位分析，同时开辟了标记诸如细胞内膜等通常无法用细胞化学标记物标记的膜的可能性，且不会产生与膜分离程序相关的潜在扭曲。
