An Investigation of Receptor-Mediated Endocytosis and Endosomal Sorting of Albumin and Transferrin in Retinal Vascular Endothelial Cells

Confluent monolayers of cultured retinal microvascular endothelial cells (RVEC's) were exposed simultaneously to two different protein-gold conjugates to determine if partitioning or sorting of protein ligands occurs during receptor mediated endocytosis in CNS microvascular cells. A mixture of bovine serum albumin (BSA), conjugated with 5nm colloidal gold (BSA-gold) and transferrin (TO, conjugated with 15nm gold (Tf-gold) were added to RVEC's and the monolayers processed, in situ, for TEM. At 0 mins the gold conjugates bound diffusely to the apical plasma membrane, but after 2 mins, both ligands were observed to cluster in clathrin-coated pits prior to internalisation in coated vesicles. At 5 mins the gold-ligands were co-localised in coated vesicles and early endosomes. After 10 mins the probes were observed in larger late endosomes where segregation was first noted and by 30mins, the mixture of gold particles appeared partitioned within late endosomes, with BSA-gold localised at the periphery and Tf-gol...