Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii
Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii
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位于活弓形虫内膜复合体管腔小叶中的筏微区
DOI：
10.1016/j.ejcb.2020.151149
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发表时间：
2021
期刊：
影响因子：
6.6
通讯作者：
Konishi R et al.
中科院分区：
文献类型：
--
作者：
Kato Yukinari;Takei Junko;Sano Masato;Asano Teizo;Sayama Yusuke;Uchida Kazuyuki;Nakagawa Takayuki;Kaneko Mika K.;Konishi R et al.
在哺乳动物细胞中，膜微域或木筏、质膜中富含甾醇和鞘磷脂的微域已被广泛研究。最近，人们发现木筏可以在包括弓形虫在内的多种寄生虫中发挥毒力作用。然而，在纳米尺度上研究脂质分子的二维分布一直是困难的。我们试图确定糖鞘糖脂GM1和GM3在Thet中的分布。在本研究中，采用快速冷冻和冷冻断裂的免疫电子显微镜方法。这种方法在物理上稳定了原位的分子，最大限度地减少了人工破坏的可能性。在内膜复合体(IMC)INT的外质(或腔)小叶中观察到GM3的标记，而在内膜复合体(IMC)INT的细胞质小叶中未观察到GM1的标记。弓形虫感染人包皮成纤维细胞-1(HFF-1)。在THET的任何传单中都没有检测到标记。性浆体膜。与感染HFF-1、弓形虫的小鼠成纤维细胞相比，尽管GM_1的S金标记密度很低，但在IMC腔小叶中同时检测到GM_1和GM_3的标记。同样的冷冻断裂EM法显示，GM1和GM3均在MF质膜的外质叶中表达。然而，在HFF-1质膜的外质叶中只检测到GM3的标记，而不检测到GM1的标记。这些结果表明，定位于IMC的GM1或GM3来自感染宿主哺乳动物细胞的质膜。此外，微域或木筏在细胞内受限空间的IMC细胞器的管腔小叶中的定位也很常见。贡迪斯揭示了木筏的一个新特点。
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, includingToxoplasma 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 theT. gondiicell 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) inT. gondiiinfected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of theT. gondiiplasma membrane. In contrast to HFF-1,T. gondiiinfected 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 ofT. gondiisuggests a novel characteristic of rafts.