The nuclear pore complex (NPC), the sole gateway for nucleocytoplasmic exchange in eukaryotic cells, allows for the passive diffusion of small molecules and transport-receptor-facilitated translocation of signal-dependent cargo molecules. Whether small molecules passively diffuse through a single central channel or through multiple holes of a hydrogel network is a subject of debate. Additionally, whether the passive and facilitated transport systems occupy distinct or overlapping physical regions of the NPC remains unclear. Here, we directly test these models using threedimensional super-resolution fluorescence microscopy of human cells. This approach reveals that a single viscous central channel in the NPC acts as the sole pathway for passive diffusion of various small molecules; transport receptors and their cargo complexes take distinct transport routes in the periphery, which is occluded by phenylalanine-glycine filaments. Furthermore, the passive and facilitated passageways in the NPC are closely correlated, and their conformations can be simultaneously regulated by Importin β1 (a major transport receptor) and RanGTP (a critical regulator of transport directionality). These results strongly favor a self-regulated viscous channel configuration in native NPCs over the porous hydrogel meshwork model. 3D imaging | nucleoporins | single-molecule fluorescence | super-resolution microscopy T he nuclear pore complex (NPC) that is embedded in the nuclear envelope (NE) functions as a highly selective gateway for an exchange of macromolecules between the cytoplasm and nucleus of eukaryotic cells. The NPC is a large assembly of approximately 30 different proteins, known as nucleoporins (Nups), with each present in an integer multiple of eight copies (1-3). Approximately one-third of the total Nups possess a "natively unfolded" structure with domains that are rich in phenylalanineglycine (FG) repeats. These FG-Nups form the selective permeability barrier in the NPC that allows for two transport modes: passive diffusion of small molecules (<40-60 kDa) and transport-receptor-facilitated translocation of cargo molecules containing specific signals (4, 5). The existence of facilitated translocation suggests that a cargo molecule would be repelled by the NPC unless it is chaperoned by transport receptors (6-8).The dissociation and association of transport-receptor-cargo complexes are guided by a concentration gradient of RanGTP (or RanGDP) across the NE (9, 10). In contrast, the passive diffusion through the NPC requires no consumption of chemical energy.Electron microscopy studies have revealed that the central nuclear pore is approximately 40-90 nm in length, with a minimum internal diameter of around 40-75 nm and an external diameter of approximately 120 nm. Flexible filaments extend approximately 50 nm into the cytoplasm, and a basket structure extends approximately 75 nm into the nucleus (5, 11). Thus, a transiting substrate can potentially interact continuously with Nups over a distance spanning approximately 200 nm. N...

No abstract

A selective barrier formed by intrinsically disordered Phe-Gly (FG) nucleoporins (Nups) allows transport receptor (TR)-facilitated translocation of signal-dependent cargos through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the configuration of the FG-Nup barrier and its interactions with multiple TRs in native NPCs remain obscure. Here, we mapped the interaction sites of various TRs or FG segments within the FG-Nup barrier by using high-speed super-resolution microscopy and used these sites to reconstruct the three-dimensional tomography of the native barrier in the NPC. We found that each TR possesses a unique interaction zone within the FG-Nup barrier and that two major TRs, importin β1 and Crm1, outcompete other TRs in binding FG Nups. Moreover, TRs may alter the tomography of the FG-Nup barrier and affect one another’s pathways under circumstances of heavy competition.