Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy Techniques

Background:

• The nuclear envelope (NE) regulates molecular transport between the nucleus and cytoplasm via nuclear pore complexes (NPCs).
• NPCs are crucial for gene expression regulation through macromolecular transport.
• The nuclear lamina, interacting with NPCs, provides structural support to the NE.

Purpose of the Study:

• To visualize the 3D structure of individual NPCs using atomic force microscopy (AFM).
• To investigate the localization of antibodies against NPC proteins (gp62) and nuclear lamina components (Xenopus lamin LIII).
• To assess the capability of AFM in identifying macromolecules associated with the NE.

Main Methods:

• Atomic Force Microscopy (AFM) for high-resolution 3D imaging of NPCs.
• Immunolabeling with primary antibodies against gp62 and Xenopus lamin LIII.
• Detection using 12-nm gold-labeled secondary antibodies and Transmission Electron Microscopy (TEM).

Main Results:

• AFM successfully visualized the 3D structure of individual NPCs.
• Anti-gp62 antibodies were localized on NPCs, and anti-LIII antibodies between NPCs using TEM.
• AFM detected secondary antibodies as 7 nm high particles on NPCs and between NPCs, correlating with TEM findings.

Conclusions:

• AFM is a viable technique for visualizing macromolecular associations with biomembranes, including NPCs.
• The study confirmed the localization of specific antibodies to NPC and nuclear lamina structures.
• AFM provides quantitative data on the size and volume of antibody-gold conjugates.