Barrier Dynamics of Nuclear Pore Complexes and Biomimetic Nanopores

Nuclear pore complexes (NPCs) mediate macromolecular traffic between the cytoplasm and the nucleus in eukaryotic cells. Tethered within each ~60 nm-diameter NPC lie numerous intrinsically disordered proteins that bear phenylalanine-glycine (FG) repeats known as FG nucleoporins (FG Nups). The FG Nups establish a selective barrier that impedes the passage of non-specific cargoes but rapidly yields to cargo-carrying transport receptors. However, the basic functional form of the FG Nups remains unresolved with respect to their spatiotemporal behaviour inside native NPCs. Here, we use high-speed atomic force microscopy (HS-AFM) to visualize nanoscopic FG Nup behaviour inside Xenopus laevis oocyte NPCs at near transport-relevant timescales. Our results show that the NPC channel is circumscribed by highly flexible, dynamically fluctuating FG Nups that elongate and retract in a stochastic manner consistent with the diffusive motion of tethered polypeptide chains. On this basis, extended FG Nups can momentarily interlink or coalesce into short-lived metastable condensates in the central channel, but do not cohere into a static meshwork that spans the entire pore. By resolving the time-dependent behaviour of FG Nups in the NPC, our findings bring consensus to barrier models that mainly disagree on static interpretations of how the FG Nups are spatially arranged in the pore. Furthermore, HS-AFM has been used to study the behavior of polyethylene glycol (PEG) polymer chains tethered inside of artificial nanopores. Our data shows that longer PEG chains serve are more effective in forming a barrier in pore than short PEG polymers. This serves as a strategy to design bio-mimetic nanopores with NPC-like functionality in the future.