Mapping of nucleoporins to the center of the nuclear pore complex by post-embedding immunogold electron microscopy.

Ultrathin sections of Lowicryl K4M embedded cultured 3T3 cells, human keratinocytes and mouse/rat liver tissue were incubated with polyspecific primary antibodies against p62 and other nucleoporins followed by 10 nm gold labeled secondary antibodies. By quantitatively evaluating both cross sections and tangential sections of the NPC, we found that irrespective of the cell type antibodies predominantly bound within a radius of 25 nm around the central axis of the nuclear pore complex (NPC). Superposition of a current structural model of the NPC with the nucleoporin distribution observed by us showed that nucleoporins mapped predominatly to the controversely discussed 'central granule'. Our experimental approach was verified by mapping gp210, another nuclear pore protein, at or very close to the NPC in the perinuclear cisterna thus establishing a distribution pattern completely different from that of the nucleoporins.