Glycosylation and importins liquefy the FG barrier in live nuclear pores.

Abstract

The nuclear pore complex (NPC) regulates molecular traffic between nucleus and cytoplasm1,2. Its permeability barrier is formed by intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG-NUPs), whose physical state has long been debated3-13. 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 observations14 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 organization4,5,7,9,15 and establish a framework for understanding how cellular context tunes intrinsically disordered proteins in health and disease.

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