Morphological analysis of surface immunoglobulin biosynthesis in mouse B lymphoid cells using immunoelectron microscopy

Abstract

Immunoelectron microscopic techniques were employed to investigate the biosynthesis of surface immunoglobulin (sIg) molecules in murine splenic B lymphocytes. In addition, cryoultramicrotomy was used due to its unique ability to retain antigenicity and provide good accessibility of external immunolabeling reagents to the cellular interior. In order to induce the biosynthesis of sIg, preexisting surface molecules were first removed completely by treatment with antibody and chymotrypsin. And the cells were then allowed to regenerate the expression of sIg molecules in culture. Our results clearly indicate that cytoplasmic immunoglobulin (cIg) exists in a close association with endoplasmic reticulum, Golgi, and vesicle structures within the cytoplasm as early as 15–30 min after antibody/enzyme treatment. After approximately 60 min, intracellular cIg molecules were observed to be incorporated into the plasma membrane through the fusion of cIg containing vesicles with the surface membrane. Reexpression of sIg reaches about 50% of its original level at 6 hr and 90% of its original level at 20 hr of incubation. Both the intracellular appearance of Ig-positive structures and reexpression of sIg on the cell surface are found to be inhibited by both puromycin and tunicamycin which suggest that both protein synthesis and glycosylation are required for sIg synthesis and reexpression. To our knowledge these studies have provided the first morphological analysis of the biosynthesis of membrane glycoproteins with intact whole cells.