O-GlcNAcylation and an importin-β radial gradient keep the FG barrier liquid in live-cell nuclear pores

The nuclear pore complex (NPC) regulates the molecular traffic between nucleus and cytoplasm. Its permeability barrier is formed by intrinsically disordered proteins (IDPs) known as FG-nucleoporins (FG-NUPs), whose physical state has long been debated. Deciphering how FG-NUPs behave inside living cells is crucial for understanding how the NPC achieves selective and rapid transport. Here, by combining site-specific labelling with picosecond time-resolved fluorescence anisotropy, we reveal that FG domains exhibit nanosecond-scale, liquid-like mobility in live cells, yet undergo a liquid-to-solid transition in vitro. Experiments and coarse-grained molecular dynamics simulations further show that importin-β, a major nuclear transport receptor, and O-linked β-N-acetylglucosamine (O-GlcNAc), a key post-translational modification of FG-NUPs together stabilise the dynamic FG network. Finely balanced FG-FG interactions modulated by O-GlcNAcylation, along with FG-importin-β interactions, maintain liquidity and enrich importin-β near the NPC periphery, while extended FG domains remain in the central channel to form the transport barrier. These findings reconcile conflicting models of FG-NUP organisation and explain the recent observations in high-resolution MINFLUX studies of importin-β depletion from the pore centre in mammalian cells. Beyond resolving debates over FG-NUP behaviour, our study underscores the importance of studying IDPs in their cellular context, with broader implications for understanding IDP-related diseases, including viral infections, cancer, and neurodegenerative disorders.