| Summary: | The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC’s internal organization consists of multiple dynamic environments with different local properties.
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