Mapping paths through the nuclear pore complex

Abstract

Nuclear pore complexes (NPCs) facilitate the fast, yet highly selective, nucleocytoplasmic transport of molecules. A recent study describes a multicolour imaging approach to chart the paths for cargo molecules through the human NPC with real-time 3D visualization of nucleocytoplasmic transport events with high spatial and temporal precision.

The nuclear envelope (NE) is the defining feature of eukaryotic cells, separating the nucleus from the cytosol; transport of molecules between these two compartments occurs through nuclear pore complexes (NPCs) — specialized channels across the NE. The human NPC is a 110-MDa multi-subunit protein complex with eight-fold rotational symmetry and filaments that extend into the cytosol and nucleoplasm, formed by ~34 different proteins¹. Electron microscopy and X-ray crystallography revealed that nuclear pore proteins or nucleoporins (NUPs) are arranged into three rings: the nucleoplasmic ring, the cytoplasmic ring and the inner ring (or scaffold ring). The latter serves as a docking platform for NUPs that have phenylalanine-glycine repeat domains (FG-NUPs), creating the central channel of the pore and forming the NPC permeability barrier^1-3. The NPC permeability barrier functions similar to a sieve, to allow selective import and export of protein and RNA molecules to and from the nucleus, facilitated by a variety of nuclear transport receptors (NTRs). In this issue of Nature Cell Biology, Chowdhury, Sau and Musser⁴ examine the NPC pathways through which this traffic flows. This process has important implications as aberrant nucleocytoplasmic transport events are hallmarks of multiple human disorders⁵ and have been reported among the earliest pathophysiological events in familial and sporadic cases of amyotrophic lateral sclerosis⁶.

While small molecules and proteins (less that ~30 kDa) can transit through the NPC by passive diffusion⁷, large cargoes utilize NTRs that interact with FG-NUPs residing in the pore’s central channel. More than 1,000 proteins per second undergo facilitated transport events through an individual NPC⁸. This transport is notable not only for its high volume, but also for its selectivity. To visualize this flow, Chowdhury, Sau and Musser⁴ developed a multicolour imaging approach for three-dimensional (3D) visualization of cargo transport in unfixed permeabilized cells. The approach built on a single-molecule astigmatism imaging technique with a high temporal resolution that allowed the authors to position the NPC scaffold and diffusing cargo complexes simultaneously. The use of permeabilized cells, a well-established system in the nuclear transport field, was important for the development of this assay. NPCs became immobile following cell permeabilization, allowing the authors to stably track cargos passing through individual pores.

Specifically, Chowdhury, Sau and Musser⁴ used nucleoporin NUP96 to locate the cytoplasmic and nucleoplasmic rings of the NPC, which showed eight-fold symmetry and positioning of NUP96 that agreed well with previous studies⁹. They separately analysed two distinct cargo molecules (a short sequence element M9 fused to β-galactosidase (M9-βGal) and nuclear localization signal fused to two molecules of fluorescent protein BFP(NLS-2xBFP)), both of which are imported by karyopherin family NTRs — a sub-class of NTRs that bind the small GTPase Ran (Fig. 1a). Both cargo complexes travelled along the NPC scaffold and formed large clouds at the cytoplasmic and nucleoplasmic sides of the NPC, with an overall hourglass-shaped distribution (Fig. 1b). The radial distributions of transportin:M9-βGal and importin-α/β (Impα/Impβ):NLS-2xBFP complexes were quite similar, with a strong preference for transport events near the NPC scaffold. The authors speculated that cargo clouds beyond the central channel reflect interactions with intrinsically disordered FG repeats of NUPs that may significantly extend into the cytosol and the nucleus. Models have been advanced in which the NPC permeability barrier is primarily determined by the presence of FG-NUPs (FG-centric model) versus contribution of crowding due to other molecules binding these nucleoporins (Kap-centric model)¹⁰. Chowdhury, Sau and Musser⁴ interpret their data in opposition to Kap-centric models. Notably, it is difficult to assess the extent to which cell permeabilization has fully depleted cargo complexes that were associated to NPCs in intact cells, and so additional characterizations will be required to fully address this question. Moreover, it remains to be investigated how active transport events and distribution of cargo complexes with NTRs may change in growing cells with active metabolism or in energy-depleted starving cells with altered NPC conformation¹¹.

Fig. 1 ∣. NTRs distribution within the NPC.

a, FG-NUPs (orange) form a dense barrier in the central channel of the NPC and around the NPC scaffold (blue). NTRs help large cargoes to pass this barrier through interactions with FG-NUPs. Transport occurs in the presence of a gradient of the small GTPase Ran, which is required for dissociation of import complexes from the cargo in the nucleus and formation of export complexes. The gradient is achieved by asymmetric localization of the Ran GTPase-activating protein RanGAP within the cytoplasm and Ran guanine exchange factor RCC1 (or RanGEF) on chromatin^13,14. The RanGTP/GDP gradient defines the directionality of transport. b, Parameters of the NPC obtained with 3D astigmatism imaging technique based on monomeric enhanced green fluorescent protein (meGFP)-tagged NUP96 localization within the NPC⁴. Position of NUP96 is aligned with transportin and Impα/Impβ pathways. The distribution of NTRs follows an hourglass shape. The colour scale represents the density of cargo complexes within the pore (the darker colour represents a higher number of localizations per volume).

Although the paths of transportin:M9-βGal and Impα/Impβ:NLS-2xBFP were similar in this system, there are about 30 different NTRs in human cells¹²; and at steady state, thousands of NTRs are passing through any given pore. It will be important to build on the current study to understand whether other NTRs share comparable transport routes or use alternative routes. Particularly if they share the same routes, it will be of interest to assess the hierarchy through which different transport complexes interact with each other and jostle for space if the capacity of the transport channel is limited. Another potential research avenue will be to assess whether these pathways may be separately controlled to up- or downregulate the passage of certain cargos in response to cellular cues while leaving the transport of other cargo molecules unperturbed. Interestingly, Chowdhury, Sau and Musser⁴ found that cargo complexes can be rejected at the midplane of the NPC, raising the intriguing question of whether FG-NUPs can search for cargo complexes and perform a ‘preselection’ at the NPC periphery to abort improperly folded cargo complexes before they reach the NPC midplane.

In summary, the work by Chowdhury, Sau and Musser⁴ describes a super-resolution approach of imaging cargo translocation during nucleocytoplasmic transport. This work offers an intriguing view into cargo-complex distribution along the NPC. This system can be applied to compare the paths of various NTRs and cargoes, as well as transport of trapped or misfolded cargo complexes, allowing a fuller view of traffic at this busy cellular transport hub.