Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA : colloidal gold by Hep G2 cells

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-la beled BSA (E6(125)I-BSA) in vivo in immature female rat liver from the plas ma membrane (P3 fraction) to the mitochondria and/or lysosomes (P2 fraction ). To further investigate this unique effect, current experiments have invo lved the use of 1,3,5(10)-estratrien-3, 17 beta -diol 17-hemisuccinate: I-1 25- BSA (E17(125)I-BSA) to demonstrate the presence of binding sites and tr anslocation of the ligand in human hepatoblastoma (Hep G2) cells. In additi on, an estrogen-BSA:colloidal gold conjugate, E17 BSA:Au, was used to direc tly visualize this uptake in Hep G2 cells. Hep G2 cells displayed high-affi nity, stereospecific binding of E17(125)I-BSA, This same ligand was also tr anslocated from the P3 fraction to the P2 fraction. In contrast,I-125-BSA w as minimally removed from the culture medium. Electron micrographs of Hep G 2 cells labeled with E17 BSA:Au demonstrated uptake of this ligand by clath rin-coated pits, indicative of receptor-mediated endocytosis. Furthermore, this ligand was also found in larger vesicles and multi-vesicular bodies, s uggesting the involvement of the compartment of uncoupling of receptor and ligand (CURL), but never in the nucleus. As early as 30 min postexposure, t he ligand could be viewed in organelles, many of which had vesiculated inte riors, resembling rounded, vesiculated mitochondria. labeled BSA was detect ed mainly in the extracellular compartment, with few multivesicular bodies containing the labeled BSA. The translocation of E17 BSA:Au was virtually e liminated by 100 nM unlabeled E17 BSA or free 17 beta -estradiol, but not 1 7 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 e ndocytosis; namely, the shuttling of estrogens to the mitochondria, in addi tion to the classical lysosomal pathway.