Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA:colloidal gold by Hep G2 cells
Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA:colloidal gold by Hep G2 cells
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DOI：
10.1677/joe.0.1660631
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发表时间：
2000-09-01
影响因子：
4
通讯作者：
Ramirez, VD
中科院分区：
文献类型：
--
作者：
Moats, RK;Ramirez, VD
以前，我们已经确定了1，3，5（10）-雌三烯-3，17 β-二醇6-（O-羧甲基）肟：I-125标记的BSA（E6（125）I-BSA）在体内未成熟雌性大鼠肝脏中从质膜（P3组分）到线粒体和/或溶酶体（P2组分）的膜介导的摄取和易位。为了进一步研究这种独特的作用，目前的实验涉及使用1，3，5（10）-雌三烯-3，17 β-二醇17-半琥珀酸酯：I-125- BSA（E17（125）I-BSA）来证明在人肝母细胞瘤（Hep G2）细胞中存在结合位点和配体易位。此外，雌激素-BSA：胶体金偶联物，E17 BSA：Au，用于直接可视化Hep G2细胞中的这种摄取。Hep G2细胞与E17（125）I-BSA的结合具有高亲和力和立体特异性。E17（125）I-BSA也可从P3部分移位到P2部分。相比之下，I-125-BSA从培养基中去除最少。用E17 BSA：Au标记的Hep G2细胞的电子显微镜照片证明了这种配体被网格蛋白包被的凹坑摄取，表明受体介导的内吞作用。此外，这种配体也被发现在较大的囊泡和多囊泡体，这表明受体和配体的解偶联（CURL）的隔间的参与，但从来没有在核中。早在30分钟后，可以看到的配体的细胞器，其中许多囊泡内部，类似圆形，囊泡状线粒体。标记的BSA主要在细胞外室中被检测到，含有标记的BSA的多泡体很少。E17 BSA：Au的易位实际上被100 nM未标记的E17 BSA或游离的17 β-雌二醇消除，但不被17 α-E6 BSA、17 α-雌二醇或P6 BSA消除，也不被细胞暴露于降低的温度消除.这些实验是第一个直观地证明膜结合和含雌激素的配体的特异性摄取，同时允许细胞内结构负责被看到。此外，他们确定了一个潜在的新途径受体介导的内吞作用，即，穿梭的雌激素的线粒体，除了经典的溶酶体途径。
Previously, we have identified a membrane-mediated uptake and translocation of 1,3,5(10)-estratrien-3, 17 beta -diol 6-(O-carboxymethyl)oxime:I-125-labeled BSA (E6(125)I-BSA) in vivo in immature female rat liver from the plasma membrane (P3 fraction) to the mitochondria and/or lysosomes (P2 fraction). To further investigate this unique effect, current experiments have involved the use of 1,3,5(10)-estratrien-3, 17 beta -diol 17-hemisuccinate: I-125- BSA (E17(125)I-BSA) to demonstrate the presence of binding sites and translocation of the ligand in human hepatoblastoma (Hep G2) cells. In addition, an estrogen-BSA:colloidal gold conjugate, E17 BSA:Au, was used to directly visualize this uptake in Hep G2 cells. Hep G2 cells displayed high-affinity, stereospecific binding of E17(125)I-BSA, This same ligand was also translocated from the P3 fraction to the P2 fraction. In contrast,I-125-BSA was minimally removed from the culture medium. Electron micrographs of Hep G2 cells labeled with E17 BSA:Au demonstrated uptake of this ligand by clathrin-coated pits, indicative of receptor-mediated endocytosis. Furthermore, this ligand was also found in larger vesicles and multi-vesicular bodies, suggesting the involvement of the compartment of uncoupling of receptor and ligand (CURL), but never in the nucleus. As early as 30 min postexposure, the ligand could be viewed in organelles, many of which had vesiculated interiors, resembling rounded, vesiculated mitochondria. labeled BSA was detected mainly in the extracellular compartment, with few multivesicular bodies containing the labeled BSA. The translocation of E17 BSA:Au was virtually eliminated by 100 nM unlabeled E17 BSA or free 17 beta -estradiol, but not 17 alpha -E6 BSA, 17 alpha -estradiol or P6 BSA, and also by exposure of the cells to reduced temperature. These experiments are the first to visually demonstrate membrane binding and specific uptake of an estrogen-containing ligand while allowing the intracellular structures responsible to be seen. Furthermore, they identify a potentially new pathway of receptor-mediated endocytosis; namely, the shuttling of estrogens to the mitochondria, in addition to the classical lysosomal pathway.