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.
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We thank M. Rexach (University of California, Santa Cruz), S. Musser (Texas A&M University) and D. Gorlich (Max Planck Institute) for providing plasmids. The project was supported by grants from the US National Institutes of Health (NIH GM094041, GM097037 and GM116204 to W.Y.).
The authors declare no competing financial interests.
Integrated supplementary information
(A) Illumination of a single NPC at the equatorial plane of a HeLa cell nucleus in the focal plane (~ 300 nm in depth between the double light blue lines) by SPEED microscopy. Single transiting molecules (red dots) through a single GFP-NPC (blue) are imaged using an inclined illumination point spread function (iPSF). C, cytoplasm; N, nucleus. (B) iPSF forms an angle of 45° to the z direction after shifted off the center in a distance of ~237 μm by a micrometer stage. 488-nm and 633-nm lasers are used to image the single NPC and the single transiting molecules through the NPC respectively, after regulated by an optical chopper to have an on-off excitation mode. The off time is at least ten folds longer than the photobleaching time of the fluorophore labeled on the targeted molecules. The figures have been adapted from previous publications with permissions (Ma, J. & Yang, W., Proc. Natl. Acad. Sci. USA 107, 7305 (2010); Ma, J. et al., Proc. Natl Acad Sci USA 109, 7326 (2012); and Ma, J. et al., Nat. Commun. 4, 2414 (2013).).
All the determined locations for each probe are within a rectangular area of 240 × 160 nm around the centroid of the NPC.
To better demonstrate the conversion process conducted onto the NPC, the nuclear pore channel could be simplified as a cylindrical channel as shown in the above figure. Our conversion process between the Cartesian and the cylindrical coordination system is to generate a virtual 3D probability density map rather than a 3D view based on 3D single-molecule tracking. In detail, electron microscopy data have revealed that the NPC has a rotationally symmetrical structure and transiting molecules through the pore driven by hydrophobic interactions or passive diffusions would have spatial symmetrical distributions at the cross-section of nuclear pore. As shown above, a high-number of randomly diffusing molecules inside the cylinder would generate rotationally symmetrical distributions at the cross-section view as that in the NPC, further resulting in an approximately uniform spatial distribution within each very small sub-region between two neighboring rings (Supplementary Fig. 3E). This uniform distribution leads that the spatial distribution along Ɵ dimension in the cylindrical system is constant. Then the 3D coordinates (R, X, Ɵ) can be simplified to be the 2D coordinates (R, X, constant). Actually, our conversion process between the Cartesian and the cylindrical systems is from 2D (X, Y) to 2D (R, X, constant). The constant Ɵ, refers to the spatial density ρ in Fig. 1E, is calculated by using the equation . (A) 3D spatial locations of randomly diffusing molecules inside a cylinder can be coordinated in a cylindrical coordination system (R, X, Ɵ). (B) The 3D molecular locations in A are projected onto a 2D plane in a Cartesian coordination system (X, Y, Z) by microscopy imaging. (C) A very thin slice (Δx) cut from the cylinder in A along x dimension. (D) The 3D spatial locations in the slice shown in C can be projected within a narrow 2D region. (E) Cross-section view of all the locations in the thin slice shown in C. These locations can be grouped into the sub-regions between concentric rings. Given the high-number randomly distributed molecules in the cylinder and the cut very thin slice, the spatial density of locations (ρi ) in each sub-region (Si ) between two neighboring rings will be rotationally symmetrical and uniform. These locations can be further projected into 1D along the Y dimension. If the locations along Y dimension are clustered in a histogram with j columns. The total number of locations in each column (Aj ) is equal to , which can be experimentally measured as shown in (Supplementary Fig. 3F).
3D probability density maps of negatively charged Nup159 (441-881) and positively charged Nsp1 (1-603) (the brighter red cloud refers the higher density) were superimposed on the NPC architecture (grey). Their spatial distributions suggest that they mainly stay in the central axial channel in the NPC. C, the cytoplasmic side; N, the nucleoplasmic side.
The comparison between the simulation result (left, adapted with permission from Ghavami, A. et al., Biophys. J. 107, 1393 (2014)) and the experimental measurement (right) of the spatial distribution of FG domains (green) in the NPC (grey) with a top view.
(A) Imp β1’s competition effect on hNup 62 (1-300). With superposition with the NPC architecture (grey), the central slice views of the 3D spatial probability maps of hNup62 (1-300) alone (left), hNup62 (1-300) at the presence of 15 μM Imp β1 (middle) and Imp β1 at the presence of 15 μM Imp β1 (right), showing the spatial distributions of hNup62 (1-300) alone (i), competing with 15-μM Imp β1 as binding FG Nups (ii) and self-competition of Imp β1 or conformational changes of FG Nups induced by 15 μM Imp β1 (iii), respectively. The major competition region between Imp β1 and hNup62 (1-300) is highlighted within a rectangular with red dashed line. Scale bar: 20 nm. (B) hNup 62 (1-300) competition effect on Imp β1. (C-D) The competitions between Imp β1 and hNup98 (1-466). (E-F) The competitions between Crm1 and hNup 62 (1-300). (G-H) The competitions between Crm1 and hNup98 (1-466).
Supplementary Figures 1–6, Supplementary Tables 1 and 2 and Supplementary Note (PDF 1658 kb)
Sequences of FG segments, Imp b1 and mutants (PDF 125 kb)
This movie shows a typical event of FG segments (here Nsp1(1-603) is used as an example) interacting with the NPC of a eukaryotic cell. Pixels are 240-nm squares, each frame was acquired in 400 μs, and the playback speed is 2500X slower than real-time. The compartment on the left side of the NPC (the green fluorescent spot) is the cytoplasm (C), and the right side is the nucleus (N). (MOV 252 kb)
This movie shows a typical import event of Imp β1 from the cytoplasm to the nucleus of a eukaryotic cell. Pixels are 240-nm squares, each frame was acquired in 400 μs, and the playback speed is 2500X slower than real-time. The compartment on the left side of the NPC (the green fluorescent spot) is the cytoplasm (C), and the right side is the nucleus (N). The Imp β1 molecule (the red fluorescent spot) starts from the cytoplasm, interacts with the NPC and ends in the nucleus. (MOV 519 kb)
This movie shows a typical export event of Crm1 from the nucleus to the cytoplasm of a eukaryotic cell. Pixels are 240-nm squares, each frame was acquired in 400 μs, and the playback speed is 2500X slower than real-time. The compartment on the left side of the NPC (the green fluorescent spot) is the cytoplasm (C), and the right side is the nucleus (N). The Crm1 molecule (the red fluorescent spot) starts from the nucleus, interacts with the NPC and ends in the cytoplasm. (MOV 375 kb)
Merged 3D spatial distributions of FG repeats and the passive diffusion route of 10-kDa dextran in the native NPC
This movie shows a cut-away view of the 3D spatial probability density map of all the recognized FG repeats in the NPC (red cloud; brighter color indicates higher spatial density) superimposed on the passive diffusion channel of 10-kDa dextran (red) and the NPC architecture (grey). C, the cytoplasmic side of the NPC. N, the nucleoplasmic side of the NPC. Bar: 20 nm. (MOV 8731 kb)
This movie shows a cut-away view of three 3D spatial probability density maps (green clouds) of FG repeats respectively recognized by FG segments, importins or exportins. The 3D pathway (red clouds) of 10-kDa dextran is also shown in a cut-away view as a comparison. All these 3D maps are superimposed on the NPC architecture (grey). C, the cytoplasmic side of the NPC. N, the nucleoplasmic side of the NPC. Bar: 20 nm. (MOV 10703 kb)
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Ma, J., Goryaynov, A. & Yang, W. Super-resolution 3D tomography of interactions and competition in the nuclear pore complex. Nat Struct Mol Biol 23, 239–247 (2016). https://doi.org/10.1038/nsmb.3174
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