Nuclear pore complexes (NPCs) are biological nanomachines that mediate the bidirectional traffic of macromolecules between the cytoplasm and nucleus in eukaryotic cells. This process involves numerous intrinsically disordered, barrier-forming proteins known as phenylalanine-glycine nucleoporins (FG Nups) that are tethered inside each pore. The selective barrier mechanism has so far remained unresolved because the FG Nups have eluded direct structural analysis within NPCs. Here, high-speed atomic force microscopy is used to visualize the nanoscopic spatiotemporal dynamics of FG Nups inside Xenopus laevis oocyte NPCs at timescales of ∼100 ms. Our results show that the cytoplasmic orifice is circumscribed by highly flexible, dynamically fluctuating FG Nups that rapidly elongate and retract, consistent with the diffusive motion of tethered polypeptide chains. On this basis, intermingling FG Nups exhibit transient entanglements in the central channel, but do not cohere into a tightly crosslinked meshwork. Therefore, the basic functional form of the NPC barrier is comprised of highly dynamic FG Nups that manifest as a central plug or transporter when averaged in space and time.
Polymer brushes are widely used to prevent the adsorption of proteins, but the mechanisms by which they operate have remained heavily debated for many decades. We show conclusive evidence that a polymer brush can be a remarkably strong kinetic barrier towards proteins by using poly(ethylene glycol) grafted to the sidewalls of pores in 30 nm thin gold films. Despite consisting of about 90% water, the free coils seal apertures up to 100 nm entirely with respect to serum protein translocation, as monitored label-free through the plasmonic activity of the nanopores. The conclusions are further supported by atomic force microscopy and fluorescence microscopy. A theoretical model indicates that the brush undergoes a morphology transition to a sealing state when the ratio between the extension and the radius of curvature is approximately 0.8. The brush-sealed pores represent a new type of ultrathin filter with potential applications in bioanalytical systems.
ILD should be evaluated carefully in all cases of JDM regardless of respiratory symptoms. CSA is a choice for steroid-resistant cases of JDM-associated ILD.
Control
of molecular translocation through nanoscale apertures
is of great interest for DNA sequencing, biomolecular filters, and
new platforms for single molecule analysis. However, methods for controlling
the permeability of nanopores are very limited. Here, we show how
nanopores functionalized with poly(ethylene glycol) brushes, which
fully prevent protein translocation, can be reversibly gated to an
“open” state by binding of single IgG antibodies that
disrupt the macromolecular barrier. On the basis of surface plasmon
resonance data we propose a two-state model describing the antibody–polymer
interaction kinetics. Reversibly (weakly) bound antibodies decrease
the protein exclusion height while irreversibly (strongly) bound antibodies
do not. Our results are further supported by fluorescence readout
from pore arrays and high-speed atomic force microscopy on single
pores. This type of dynamic barrier control on the nanoscale provides
new possibilities for biomolecular separation and analysis.
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