Anti-immunoglobulin (Ig) coupled to ferritin or hemocyanin was used to map the distribution of Ig molecules on lymphocytes derived from bone marrow (B lymphocytes) by freeze-etching. The labeled anti-Ig was distributed all over the membrane in the form of random interconnected patches forming a lacy, continuous network. This was the pattern of lymphocytes labeled at 4°C with the anti-Ig. After warming at 37°C, the labeled molecules concentrated into a single area of the cell (forming the cap) and were rapidly internalized in small vesicles Freeze-etching showed close packing of the labeled molecules in the cap area. There was evidence that in the cap area the Ig molecules were exfoliated from the plane of the membrane, suggesting that the Ig may be superficial to the bilipid layer, or weakly anchored to the membrane.
Similar studies were made using antibodies to histocompatibility antigens. Thymocytes were labeled with anti-H-2 and ferritin anti-Ig at 4°C. Freeze-etching showed large patches scattered over the membrane and separated from each other by several thousand angstroms. This distribution may, in part, explain why H-2 antigens do not readily form a cap; the large patches are beyond the reach of even a double ligand (sandwich) reaction.
The antigens that reacted with heterologous anti-lymphocyte globulin (ALG) were found in small noninterconnected clusters a few hundred angstroms apart. Such clusters presumably cannot be linked by a single antibody but can by a sandwich (ligand to ligand-antigen) reaction. In previous studies it was found that ALG antigens form a cap only after a sandwich reaction. Finally, the receptors for concanavalin A (Con A) were found in a lacy, irregular interconnected, random network. The spatial distribution of these moieties on the membrane may, in great part, determine their movement after reaction with one or two ligands.