Intracellular Distribution of a Biotin-labeled Ganglioside, GM1, by Immunoelectron Microscopy After Endocytosis in Fibroblasts

Abstract

A radioactive and biotin-labeled analogue of GM1 (biotin-GM1) was synthesized which enabled us to analyze its intracellular distribution in the compartments of the endocytic route by electron microscopic immunocytochemistry using thin sections of human skin fibroblasts labeled with gold-conjugated antibiotin antibodies. Metabolic studies with the biotin-GM1 showed its partial degradation to the corresponding GM2 and GM3 derivatives. Further degradation was inhibited by the biotin residue. The distribution of biotin-GM1 after uptake by cells was studied by postembedding labeling techniques. On the plasma membrane the biotin-GM1 was detectable in the form of patches (0.1 μm in diameter), in caveola-like structures and, to a much lesser extent, in coated pits or vesicles. During endocytic uptake, the biotin-GM1 became detectable in organelles identified as late endosomes and lysosomes. The intracellular distribution of the biotin-GM1 was compared to the localization of the EGF receptor in EGF-stimulated fibroblasts. Both the biotin-GM1 and the EGF receptor were transported to intraendosomal and intralysosomal membranes, indicating that both membrane constituents follow the same pathway of endocytosis. Our observations show that biotin-GM1 can be successfully incorporated into the plasma membrane and be used as a tool for morphological detection of its pathway to lysosomes. (J Histochem Cytochem 47:1005–1014, 1999)