Phenylalanine-glycine nucleoporins (FG Nups) are intrinsically disordered proteins that generate the permeability barrier within nuclear pore complexes (NPCs). NPCs are remarkable sorting machines that mediate nucleocytoplasmic transport (NCT) in eukaryotic cells. On one hand, NCT is rapid and selective for cargo-carrying nuclear transport receptors termed karyopherins (Kaps). On the other, the permeability barrier obstructs the passage of non-specific cargoes. Importantly, NPC function is underscored by, amongst others, neurodegenerative disorders and viral pathogenesis that are linked to FG Nup/Kap dysfunction. Despite being central to NPC function, we do not understand the spatiotemporal behavior of the FG Nups in the NPC and the permeability barrier remains highly debated. Due to their conformational flexibility, a structural characterization of the permeability barrier remains lacking and lags significantly behind advances in our understanding of NPC scaffold structure. Likewise, it remains unclear how Kap-cargo interactions with the FG Nups might alter the behavior of the permeability barrier to traverse the NPC. This is further related to the question of whether the permeability barrier plays a role in influencing large-scale conformational changes in the NPC such as to accommodate large cargoes. In this work, we will tackle these two major themes: (i) FG Nup dynamics within the NPC permeability barrier; and (ii) to explore its links to conformational changes in the NPC. To do so, we will employ high-speed atomic force microscopy (HS-AFM) to investigate the permeability barrier within NPCs isolated from S. cerevisiae (budding yeast) nuclei at the single NPC level, at transport-relevant length scales (nm) and timescales (~100 ms). Specifically we will characterize FG Nup dynamic behavior in the absence and presence of Kap-cargo complexes in both native NPCs and ÄFG mutant NPCs. In addition, we will evaluate how the permeability barrier might act as a mechanosensor that induces large-scale conformational changes in the NPC. This will involve a systematic study using different ÄFG mutant NPCs that exhibit different degrees of FG Nup cross-linking within their respective permeability barriers. On this basis, we hypothesize that disrupting inter-FG Nup interactions (e.g., by FG-domain deletions, amphipathic alcohols, large cargo complexes, etc.) facilitates pore dilation by reducing the amount of tension imposed by the FG Nups on the NPC scaffold. Finally, we will substantiate our NPC-level findings at the individual FG Nup-level by investigating the permeability barrier generated by FG Nups tethered within artificial nanochannels (also termed NPC mimics).