Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• The nuclear pore complex (NPC) is a massive cellular machine regulating transport between the nucleus and cytoplasm.
• Understanding NPC structure and function is crucial, with recent advances in cryo-electron microscopy and super-resolution imaging.
• Direct visualization of single NPC proteins has remained a significant challenge.

Purpose of the Study:

• To develop a method for visualizing single copies of nuclear pore complex proteins in human cells.
• To achieve high-resolution 3D imaging of nucleoporins at the single-molecule level.
• To enable the study of multicomponent protein complexes within the NPC.

Main Methods:

• Genetically encoded self-labeling enzymes (SNAP-tag, HaloTag) were employed for protein labeling.
• DNA-PAINT super-resolution microscopy was utilized for high-precision imaging.
• Three-dimensional reconstruction of labeled nucleoporins was performed.

Main Results:

• Single copies of nucleoporins within the human Y-complex were resolved in 3D.
• Imaging achieved a precision of approximately 3 nanometers.
• The study demonstrated the feasibility of studying protein complexes at the single-protein level using optical microscopy.

Conclusions:

• The combination of self-labeling enzymes and DNA-PAINT microscopy enables unprecedented visualization of single NPC proteins.
• This technique opens new avenues for dissecting the molecular architecture and dynamics of large protein assemblies.
• Future studies can leverage this method to investigate nucleocytoplasmic transport mechanisms with single-protein resolution.