title: Validation of the biotinyl ligand-avidin-gold technique.
abstract: We demonstrate here that the intracellular routing of biotinylated ligands was not affected by the attachment of streptavidin gold colloids so long as the electron-dense marker was added after the biotinyl ligand-receptor interaction had occurred. The binding, internalization, and intracellular routing of three different biotinyl ligands were followed in mouse LM fibroblasts. The biotinyl (B) ligands included B-choleragenoid (B-CTd), B-wheat germ agglutinin (B-WGA), and B-Pseudomonas exotoxin A (B-PE). All three ligands showed distinct intracellular trafficking patterns. B-WGA and B-PE entered via clathrin-coated pits, whereas B-CTd did not. After entry, B-CTd was routed to the lysosomal compartment without involvement of the Golgi. Although B-PE and B-WGA were also routed to the lysosomal compartment, a significant portion of these two ligands was observed in association with the Golgi. B-WGA, however, remained in the endosomal and Golgi compartments longer than did B-PE. We also monitored the internalization and routing of native PE by an indirect immunoperoxidase technique done in conjunction with saponin solubilization. The results corroborated the observations with the biotinyl-PE-streptavidin-gold method. In contrast, biotinyl-PE added to streptavidin-gold before addition to LM cells was poorly internalized and routed aberrantly. From these observations we conclude that the biotinyl ligand-avidin-gold technique is a valid method for following the binding, internalization, and intracellular routing of ligands.
authors.affiliation: Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, Ohio 45267-0521.
authors.forename: R E
authors.initials: RE
authors.lastname: Morris
authors.forename: G M
authors.initials: GM
authors.lastname: Ciraolo
authors.forename: C B
authors.initials: CB
authors.lastname: Saelinger
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001360:Avidin / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism*; D002460:Cell Line; D002772:Cholera Toxin / Q000378:metabolism*; D004705:Endocytosis; D005098:Exotoxins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D037521:Virulence Factors; D014909:Wheat Germ Agglutinins / Q000378:metabolism*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001360:Avidin; D001427:Bacterial Toxins; D001710:Biotin; D002772:Cholera Toxin; D005098:Exotoxins; D037521:Virulence Factors; D014909:Wheat Germ Agglutinins; D000097668:Pseudomonas aeruginosa Exotoxin A