Self-regulated viscous channel in the nuclear pore complex

Abstract

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...

Distinct, but not completely separate spatial transport routes in the nuclear pore complex

“…3). 79,82 Recently, the distribution of single-molecule locations along the θ dimension in each NPC was experimentally confirmed to be approximately homogenous.…”

“…To meet the above requirements for the mapping of nuclear transport, an innovative imaging approach that combines single-particle tracking and a deconvolution algorithm has recently been developed in our laboratory. [79][80][81][82] not the θ coordinate. The distribution of single-molecule locations along the θ dimension is then averaged to make it uniform, and thus, θ becomes a constant for each single-molecule location, as (r, x, constant).…”

“…79 Next, a second laser with a different wavelength following the same beam path as the 488-nm laser is used to conduct single-particle tracking of labeled in-transit molecules through this illuminated NPC. 79 Noteworthy, the major difference between SPEED microscopy and a previous reported highly inclined and laminated optical sheet microscopy is the different illumination volume formed at the focal plane of objective. 90 The former provides a diffraction-limit point excitation and the latter generates a plane illumination.…”

“…[79][80][81][82] (1) Single NPCs in cells can be First, single-particle tracking of in-transit molecules through a single NPC is realized using single-point edge-excitation sub-diffraction (SPEED) microscopy. The first goal of SPEED microscopy is to illuminate a single NPC in the NE in permeabilized and living cells.…”

Nuclear export, enlightened

“…Are there protein spurs (side-chains or, at greater reach, N-or C-termini) that emanate inward acting as a rheological barrier or are they active players (i.e., transport as a "spaghetti opera")? This remains a controversial aspect of the nuclear export field, and a recent study has suggested a very different biophysical structure of the NPC central channel (15). Single-particle tracking could play a role in the further resolution of this issue, combined with the appropriate experimental variables.…”