IMMUNOLOCALIZATION OF LAMINS AND NUCLEAR-PORE COMPLEX PROTEINS BY ATOMIC-FORCE MICROSCOPY

The nuclear envelope functions as a selective barrier separating the n uclear from the cytosolic compartment. Nuclear pore complexes (NPCs) m ediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied ato mic force microscopy (AFM) to visualize the three dimensional (3D) str ucture of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another di rected against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold -labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antib odies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on N Es. According to the 3D images, the volume of a single gold particle c onjugated with secondary antibodies was 10 203 nm(3). This volume is e quivalent to the volume of 38 IgG molecules associated with one indivi dual gold particle. A similar volume of 11 987 nm(3) was calculated fr om a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identif ying antibodies or other macromolecules associated with biomembranes.