Glycosylation and importins liquefy the FG barrier in live nuclear pores

Abstract The nuclear pore complex (NPC) regulates molecular traffic between nucleus and cytoplasm 1,2 . Its permeability barrier is formed by intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG-NUPs), whose physical state has long been debated 3–13 . Deciphering how FG-NUPs behave inside living cells is crucial for understanding how NPCs achieve both selective and rapid transport. By combining site-specific labelling with time-resolved fluorescence anisotropy, here we show that FG domains exhibit nanosecond-scale, liquid-like segmental motion in live cells, whereas reconstituted FG condensates undergo a liquid-to-solid transition in vitro. Experiments and molecular-dynamics simulations further show that importin-β, a principal nuclear transport receptor, and O-linked β- N -acetylglucosamine, a key post-translational modification of FG-NUPs, together stabilize the dynamic FG network. Finely balanced FG–FG and FG–importin-β interactions maintain FG-NUP liquidity and generate a radial organization in which importin-β is enriched near the NPC periphery and extended FG domains remain in the central channel, forming the transport barrier. This organization explains recent MINFLUX observations 14 of rim-biased transport trajectories and is supported here by direct MINFLUX imaging of importin-β in mammalian cells. Together, our results reconcile conflicting models of FG-NUP organization 4,5,7,9,15 and establish a framework for understanding how cellular context tunes intrinsically disordered proteins in health and disease.