The nuclear pore complex regulates cargo transport between the cytoplasm and the nucleus. We set out to correlate the governing biochemical interactions to the nanoscopic responses of the phenylalanineglycine (FG)–rich nucleoporin domains, which are involved in attenuating or promoting cargo translocation. We found that binding interactions with the transport receptor karyopherin-β1 caused the FG domains of the human nucleoporin Nup153 to collapse into compact molecular conformations. This effect was reversed by the action of Ran guanosine triphosphate, which returned the FG domains into a polymer brush-like, entropic barrier conformation. Similar effects were observed in Xenopus oocyte nuclei in situ. Thus, the reversible collapse of the FG domains may play an important role in regulating nucleocytoplasmic transport.

Natively unfolded phenylalanine-glycine (FG)-repeat domains are alleged to form the physical constituents of the selective barrier-gate in nuclear pore complexes during nucleocytoplasmic transport. Presently, the biophysical mechanism behind the selective gate remains speculative because of a lack of information regarding the nanomechanical properties of the FG domains. In this work, we have applied the atomic force microscope to measure the mechanical response of individual and clusters of FG molecules. Single-molecule force spectroscopy reveals that FG molecules are unfolded and highly flexible. To provide insight into the selective gating mechanism, an experimental platform has been constructed to study the collective behavior of surface-tethered FG molecules at the nanoscale. Measurements indicate that the collective behavior of such FG molecules gives rise to an exponentially decaying long-range steric repulsive force. This finding indicates that the molecules are thermally mobile in an extended polymer brush-like conformation. This assertion is confirmed by observing that the brush-like conformation undergoes a reversible collapse transition in less polar solvent conditions. These findings reveal how FG-repeat domains may simultaneously function as an entropic barrier and a selective trap in the near-field interaction zone of nuclear pore complexes; i.e., selective gate.

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-D-dependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolicSalmonellaor upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolicSalmonellaseconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of non-canonical inflammasome signaling.