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

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Abstract
The early events in the entry of ligands into cultured fibroblasts through plasma membrane clathrin-coated pits were examined by using serial sections and EM. Swiss 3T3 cells were labeled at 4.degree. C with concanavalin A-horseradish peroxidase and warmed to 37.degree. 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 enhances contrast of membranous structures. Serial sections taken both parallel to and perpendicular to the substratum were examined by EM, 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 .apprxeq. 170 .ANG. in width and up to 7000 .ANG. in length. These necks were often surrounded by a dense filamentous network. Evidently, isolated coated vesicles do not form during the endocytosis process in Swiss 3T3 cells.