Investigating molecular crowding within nuclear pores using polarization-PALM

Area of Science:

• Cell Biology
• Biophysics
• Molecular Mechanisms

Background:

• The nuclear pore complex (NPC) regulates transport between the nucleus and cytoplasm.
• Its function depends on intrinsically disordered phenylalanine-glycine (FG) polypeptide assemblies.
• Characterizing FG-network dynamics and macromolecular mobility within intact NPCs is challenging.

Purpose of the Study:

• To investigate the architecture and dynamics of the FG-network within the NPC.
• To develop and apply advanced microscopy techniques for probing FG-polypeptide mobility.
• To understand the local environments and transport properties within the NPC.

Main Methods:

• Utilized a combination of fluorescence polarization, super-resolution microscopy, and mathematical analysis.
• Employed polarization PALM (p-PALM) to measure rotational mobility of fluorescent probes.
• Examined FG-polypeptide dynamics at various locations within the NPC under different conditions.

Main Results:

• Demonstrated that p-PALM effectively probes low rotational mobilities missed by bulk anisotropy methods.
• Revealed that the NPC's internal organization comprises multiple dynamic environments.
• Characterized different local properties within these dynamic environments.

Conclusions:

• p-PALM is a powerful tool for studying crowded cellular environments like the NPC.
• The NPC's FG-network exhibits complex, heterogeneous dynamics.
• These findings advance our understanding of nucleocytoplasmic transport regulation.