Formation of receptosomes from plasma membrane coated pits during endocytosis: analysis by serial sections with improved membrane labeling and preservation techniques.

The early events in the entry of ligands into cultured fibroblasts through plasma membrane clathrin-coated pits were examined by using serial sections and electron microscopy. Swiss 3T3 cells were labeled at 4 degrees C with concanavalin A-horseradish peroxidase and warmed to 37 degrees C for 1 or 2 min to label organelles participating in endocytosis. Cells were fixed in glutaraldehyde and further preserved with a membrane contrast-enhancement technique that utilized sequential osmium/ferrocyanide, thiocarbohydrazide, and osmium/ferrocyanide treatment in situ. This technique provides exceptional preservation and enhanced contrast of membranous structures. Serial sections taken both parallel to and perpendicular to the substratum were examined by electron microscopy, and 263 clathrin-coated structures containing horseradish peroxidase were evaluated for their connection to the plasma membrane. All of these coated structures were found to be connected to the plasma membrane either directly or via necks approximately equal to 170 A in width and up to 7,000 A in length. These necks were often surrounded by a dense filamentous network. These results strongly suggest that isolated coated vesicles do not form during the endocytosis process in Swiss 3T3 cells.