Endocytosis of folate labeled proteins: Ultrastructural localization in kb cells

Abstract

Recently, methods for the delivery of macromolecules into cells via covalent coupling to vitamins have been described for plant and animal cells. This method utilizing vitamin receptor endocytosis concentrates macromolecules inside the cell in an active form that is nondegradative. The purpose of this study was to determine the specific location of bovine serum albumin-folic acid-colloidal gold (BSA-F-CG) conjugates within KB cells. Bovine serum albumin was covalently coupled to folic acid (BSA-F) and used to stabilize 15 nm colloidal gold (CG) particles. KB cells were incubated with BSA-F-CG or BSA-CG at either 11°C for 2 hours or 37°C for 15 minutes to allow binding, and the cells washed to remove unbound material. Some cells were fixed immediately, and others were incubated for additional time periods. BSA-CG particles nonspecifically pinocytosed by control cells were only found in large dense endosomes after 6 hours incubation. In cells incubated with BSA-F-CG, CG particles decorated the plasma membrane, and were found in uncoated pits. At 30 minutes CG particles could be found in multivesicular bodies (MVB’s), small vesicles, and dense endosomes. At 6 hours, CG particles were found in various dense endosomes, MVB’s, dense endosomes associated with the Golgi apparatus, and free in the cytoplasm. Cells that were pulsed with BSA-F-CG for 15 minutes at 37°C had CG particles on the surface and uncoated pits, small vesicles and MVB’s. After 60 and 360 minutes, BSA-F-CG was located in MVB’s, clear and dense endosomes, and MVB’s associated with the Golgi apparatus. Control cells incubated with BSA-CG had a few CG particles located in small clear endosomes at 15 minutes and large dense endosomes at 6 hours. For comparison, 5nm colloidal gold was stabilized with transferrin (TF) and incubated alone with KB cells or coincubated with 15 nm BSA-F-CG. The 5 nm TF-CG was localized in coated pits and on the rim of vesicular structures resembling CURL (Compartment of Uncoupling of Receptor and Ligand) at 15 minutes. There was no colocalization of TF-CG and BSA-F-CG at 15 minutes. TF-CG and BSA-F-CG were found separately and together in MVB’s at 1 hour. At six hours TF-CG and BSA-F-CG frequently colocalized in large dense endosomes. Folate labeled proteins endocytosed in this study shared some common compartments with transferrin, but there were several points of divergence. Transferrin-CG is taken up via coated pits whereas BSA-F-CG enters the cell via uncoated pits. At 15 minutes, internalized TF-CG is associated primarily with vesicles resembling CURL, and BSA-F-CG is found in MVB’s. At one hour, both TF-CG and BSA-F-CG may be found separately and together in MVB’s, and at 6 hours both may be found separately and together in dense endosomes. The BSA-F-CG particles free in the cytoplasm may be released during transport of the ligand, possibly by MVB’s that associate with the Golgi apparatus. The BSA-F-CG particles localized in this study may represent the normal location of endocytosed folate or their location could be an aberration of the normal pathway.