Phase separation of intrinsically disordered FG-Nups is driven by highly dynamic FG motifs.

The intrinsically disordered FG-Nups in the central channel of the nuclear pore complex (NPC) form a selective permeability barrier, allowing small molecules to traverse by passive diffusion, while large molecules can only translocate with the help of nuclear transport receptors. The exact phase state of the permeability barrier remains elusive. In vitro experiments have shown that some FG-Nups can undergo phase separation into condensates that display NPC-like permeability barrier properties. Here, we use molecular dynamics simulations at amino acid resolution to study the phase separation characteristics of each of the disordered FG-Nups of the yeast NPC. We find that GLFG-Nups undergo phase separation and reveal that the FG motifs act as highly dynamic hydrophobic stickers that are essential for the formation of FG-Nup condensates featuring droplet-spanning percolated networks. Additionally, we study phase separation in an FG-Nup mixture that resembles the NPC stoichiometry and observe that an NPC condensate is formed containing multiple GLFG-Nups. We find that the phase separation of this NPC condensate is also driven by FG-FG interactions, similar to the homotypic FG-Nup condensates. Based on the observed phase separation behavior, the different FG-Nups of the yeast NPC can be divided into two classes: The FG-Nups (mostly GLFG-type) located in the central channel of the NPC form a highly dynamic percolated network formed by many short-lived FG-FG interactions, while the peripheral FG-Nups (mostly FxFG-type) at the entry and exit of the NPC channel likely form an entropic brush.

Diameter dependence of transport through nuclear pore complex mimics studied using optical nanopores.

The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.