IMMUNOGOLD LABELING OF ONCOGENIC AND TUMOR RELATED PROTEINS

Abstract

Immunogold labeling electron microscopy technique has been used to study the ultrastructural localization of oncogenic proteins: Mos, Met, Ski, and the tumor-associated protein, Mud, as well as their spatial relationship with other tumor-related proteins. By pre- and postembedding immunogold labeling electron microscopy techniques, it was demonstrated that the Mos protein pp39ᵐᵒˢ colocalized with microtubule bundles, suggesting that microtubulin or microtubule-associated protein(s) may be the substrate of Mos. Met protein was labeled at the microvilli of the lumen that are formed in a cultured mammary T47D tumor cell lines, suggesting its potential involvement in lumen formation. Ski localization experiments revealed a unique globular structure-"Ski body"-that is present inside the nucleus of interphase chicken embryo fibroblast infected with Ski cDNA FB29 and FB2-29. Ski bodies were also found scattered in the cytoplasm of metaphase FB29 and FB2-29 Ski expressing chicken embryo fibroblasts. In T47D cells, tumor-associated protein Mud was associated with both the plasma membrane and the membranes of secretory vesicles in the cytoplasm. In MUC1 transfected NIH3T3 cells, however, labeling showed that, in addition to the plasma membrane and the membranes of secretory vesicles, some Muc1 gold spheres were detected inside the secretory vesicles, suggesting that the subcellular localization of the protein may vary in different cell types.