Immunoelectron Microscopy on Receptor and Ligand Sorting Sites
While several methods are available for characterizing ligand localization (e. g. labeling with radioactive or electrondense tracers), receptor molecules themselves can only be localized in situ by immunocytochemical means. The increasing need for precise subcellular detection has coincided with recent developments of powerful immunoelectron microscope techniques. Of these we have chosen a method which relies on ultrathin cryosections and labeling with colloidal gold particles . Double-labeling can easily be achieved using gold preparations of different sizes . Immuno double-labeling is of special value when different receptors and ligands are studied simultaneously. Using this method we have directly compared the endocytotic pathways of the receptors for asialoglycoproteins (ASGP-R), mannose 6-phosphate residues on lysosomal enzymes (MP-R) and polymeric IgA (IgA-R) in liver parenchymal cells and hepatoma cells (Hep G2). ASGP-R is exclusively present in liver cells and mediates the endocytosis of ASGP-ligand from the blood. The uptake in coated vesicles is followed by a rapid transfer through an acidic pre-lysosomal compartment to lysosomes. The low pH facilitates uncoupling of receptors and ligands. Next, receptors recirculate to the plasma membrane via an as yet unknown pathway. The MP-R on the other hand, binds to its ligand somewhere intracellularly but like ASGP-R makes use of an acidic prelysosomal compartment for ligand uncoupling. In addition, in many cell types surface MP-R is engaged in the internalization of exogenous ligand.
KeywordsLysosomal Enzyme Immunoelectron Microscopy Coated Vesicle Liver Parenchymal Cell Colloidal Gold Particle
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