Membrane microdomains or rafts, sterol- and sphingolipid-rich microdomains in the plasma membrane have been studied extensively in mammalian cells. Recently, rafts were found to mediate virulence in a variety of parasites, including Toxoplasma gondii. However, it has been difficult to examine a two-dimensional distribution of lipid molecules at a nanometer scale. We tried to determine the distribution of glycosphingolipids GM1 and GM3, putative raft components in the T. gondii cell membrane in this study, using a rapid-frozen and freeze-fractured immuno-electron microscopy method. This method physically stabilized molecules in situ, to minimize the probability of artefactual disruption. Labeling of GM3, but not GM1, was observed in the exoplasmic (or luminal), but not the cytoplasmic, leaflet of the inner membrane complex (IMC) in T. gondii infected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of the T. gondii plasma membrane. In contrast to HFF-1, T. gondii infected in mouse fibroblast (MF), labelings of both GM1 and GM3 were detected in the IMC luminal leaflet, although GM1′s gold labeling density was very low. The same freeze-fracture EM method showed that both GM1 and GM3 were expressed in the exoplasmic leaflet of the MF plasma membrane. However, labeling of only GM3, but not GM1, was detected in the exoplasmic leaflet of the HFF-1 plasma membrane. These results suggest that GM1 or GM3, localized in the IMC, is obtained from the plasma membranes of infected host mammalian cells. Furthermore, the localization of microdomains or rafts in the luminal leaflets of the intracellular confined space IMC organelle of T. gondii suggests a novel characteristic of rafts.

已经在哺乳动物细胞中广泛研究了质膜中的膜微区或筏、富含甾醇和鞘脂的微区。最近，发现筏介导多种寄生虫的毒力，包括弓形虫。然而，很难在纳米尺度上检查脂质分子的二维分布。我们试图确定鞘糖脂 GM1 和 GM3 的分布，它们是弓形虫中推定的筏组分本研究中的细胞膜，使用快速冷冻和冷冻断裂的免疫电子显微镜方法。这种方法在原位物理稳定分子，以最大限度地减少人为破坏的可能性。在感染人类包皮成纤维细胞-1 (HFF-1) 的弓形虫的内膜复合物 (IMC) 的外质（或管腔）中观察到标记 GM3，但未在细胞质中观察到 GM3，但未在细胞质中观察到。在弓形虫质膜的任何小叶中均未检测到标记。与 HFF-1 相比，刚地弓形虫在小鼠成纤维细胞 (MF) 中感染的 GM1 和 GM3 标记在 IMC 腔小叶中检测到，尽管 GM1 的金标记密度非常低。相同的冷冻断裂 EM 方法表明 GM1 和 GM3 均在 MF 质膜的外质小叶中表达。然而，在 HFF-1 质膜的外质小叶中仅检测到 GM3 的标记，而未检测到 GM1。这些结果表明，位于 IMC 中的 GM1 或 GM3 是从受感染宿主哺乳动物细胞的质膜中获得的。此外，在弓形虫的细胞内密闭空间 IMC 细胞器的腔内小叶中微域或筏的定位表明了筏的新特征。