Dynamic molecular mechanism of the nuclear pore complex permeability barrier

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport of specific macromolecules while impeding the exchange of unsolicited material. However, key aspects of this gating mechanism remain controversial. To address this issue, we determined the nanoscopic behavior of the permeability barrier directly within yeast S. cerevisiae NPCs at transport-relevant timescales. We show that the large intrinsically disordered domains of phenylalanine-glycine repeat nucleoporins (FG Nups) exhibit highly dynamic fluctuations to create transient voids in the permeability barrier that continuously shape-shift and reseal, resembling a radial polymer brush. Together with cargo-carrying transport factors the FG domains form a feature called the central plug, which is also highly dynamic. Remarkably, NPC mutants with longer FG domains show interweaving meshwork-like behavior that attenuates nucleocytoplasmic transport in vivo. Importantly, the bona fide nanoscale NPC behaviors and morphologies are not recapitulated by in vitro FG domain hydrogels. NPCs also exclude self-assembling FG domain condensates in vivo, thereby indicating that the permeability barrier is not generated by a self-assembling phase condensate, but rather is largely a polymer brush, organized by the NPC scaffold, whose dynamic gating selectivity is strongly enhanced by the presence of transport factors.</p

Nuclear Pore Membrane Proteins Self-Assemble into Nanopores

Large multiprotein nanopores remain difficult to reconstitute in vitro, such as, for instance, the nuclear pore complex (NPC) that regulates macromolecular transport between the nucleus and cytoplasm in cells. Here, we report that two NPC pore membrane proteins self-assemble into ∼20 nm diameter nanopores following in vitro reconstitution into lipid bilayers. Pore formation follows from the assembly of Pom121 and Ndc1 oligomers, which arrange into ringlike membrane structures that encircle aqueous, electrically conductive pores. This represents a key step toward reconstituting membrane-embedded NPC mimics for biological studies and biotechnological applications

High-Resolution Imaging of a Single Gliding Protofilament of Tubulins by HS-AFM

In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins

Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome α Subunits

Eukaryotic proteasomes harbor heteroheptameric &#945;-rings, each composed of seven different but homologous subunits &#945;1&#8722;&#945;7, which are correctly assembled via interactions with assembly chaperones. The human proteasome &#945;7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric &#945;6. We comprehensively characterized the oligomeric state of human proteasome &#945; subunits and demonstrated that only the &#945;7 subunit exhibits this unique, self-assembling property and that not only &#945;6 but also &#945;4 can disrupt the &#945;7 double ring. We also demonstrated that mutationally monomerized &#945;7 subunits can interact with the intrinsically monomeric &#945;4 and &#945;6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks