Imaging of single-molecule translocation through nuclear pore complexes

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Nuclear pore complexes (NPCs) regulate transport between the nucleus and cytoplasm.
• Previous studies relied on in vitro models, limiting understanding of in vivo transport dynamics.
• The role of G protein Ran in NPC transport is established, but specific cofactor-NPC interactions remain unclear.

Purpose of the Study:

• To directly monitor protein transport through NPCs in real-time using single-molecule fluorescence microscopy.
• To determine the kinetics and spatial dynamics of a model protein substrate during NPC transit.
• To identify the rate-limiting step in nuclear protein import.

Main Methods:

• Single-molecule fluorescence microscopy was employed to track a model protein substrate (NLS-2xGFP).
• Experiments were conducted in permeabilized cells to mimic physiological conditions.
• Quantitative analysis of substrate interaction time and nuclear accumulation rates was performed.

Main Results:

• The model protein substrate interacted with NPCs for an average of 10 +/- 1 ms.
• NPCs demonstrated a high transport capacity, estimated at over 10 substrate molecules simultaneously.
• Substrate molecules spent most transit time undergoing random motion within the NPC's central pore.
• The rate-limiting step for transport was identified as the substrate's escape from the central pore.

Conclusions:

• Single-molecule microscopy provides unprecedented resolution for studying NPC transport dynamics.
• NPCs possess a high throughput capacity for molecular transport.
• Substrate diffusion within the central pore and subsequent exit are critical determinants of nuclear import efficiency.