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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Rout, M.P. et al. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 (2000).
Suntharalingam, M. & Wente, S.R. Peering through the pore: nuclear pore complex structure, assembly, and function. Dev. Cell 4, 775–789 (2003).
Fried, H. & Kutay, U. Nucleocytoplasmic transport: taking an inventory. Cell. Mol. Life Sci. 60, 1659–1688 (2003).
Fahrenkrog, B. & Aebi, U. The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat. Rev. Mol. Cell Biol. 4, 757–766 (2003).
Denning, D.-P., Patel, S.S., Uversky, V., Fink, A.L. & Rexach, M. Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded. Proc. Natl. Acad. Sci. USA 100, 2450–2455 (2003).
Stewart, M. Molecular mechanism of the nuclear protein import cycle. Nat. Rev. Mol. Cell Biol. 8, 195–208 (2007).
Palmeri, D. & Malim, M.H. Importin beta can mediate the nuclear import of an arginine-rich nuclear localization signal in the absence of importin alpha. Mol. Cell. Biol. 19, 1218–1225 (1999).
Hoelz, A., Debler, E.W. & Blobel, G. The structure of the nuclear pore complex. Annu. Rev. Biochem. 80, 613–643 (2011).
Rout, M.P. & Blobel, G. Isolation of the yeast nuclear pore complex. J. Cell Biol. 123, 771–783 (1993).
Terry, L.J. & Wente, S.R. Flexible gates: dynamic topologies and functions for FG nucleoporins in nucleocytoplasmic transport. Eukaryot. Cell 8, 1814–1827 (2009).
Macara, I.G. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570–594 (2001).
Rout, M.P. & Aitchison, J.D. The nuclear pore complex as a transport machine. J. Biol. Chem. 276, 16593–16596 (2001).
Peters, R. Translocation through the nuclear pore complex: selectivity and speed by reduction-of-dimensionality. Traffic 6, 421–427 (2005).
Lim, R.Y. et al. Nanomechanical basis of selective gating by the nuclear pore complex. Science 318, 640–643 (2007).
Rout, M.P., Aitchison, J.D., Magnasco, M.O. & Chait, B.T. Virtual gating and nuclear transport: the hole picture. Trends Cell Biol. 13, 622–628 (2003).
Ribbeck, K. & Görlich, D. Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 20, 1320–1330 (2001).
Frey, S. & Görlich, D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 130, 512–523 (2007).
Mohr, D., Frey, S., Fischer, T., Güttler, T. & Görlich, D. Characterisation of the passive permeability barrier of nuclear pore complexes. EMBO J. 28, 2541–2553 (2009).
Frey, S., Richter, R.P. & Görlich, D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314, 815–817 (2006).
la Cour, T. et al. Analysis and prediction of leucine-rich nuclear export signals. Protein Eng. Des. Sel. 17, 527–536 (2004).
Yamada, J. et al. A bimodal distribution of two distinct categories of intrinsically disordered structures with separate functions in FG nucleoporins. Mol. Cell. Proteomics 9, 2205–2224 (2010).
Ma, J. & Yang, W. Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots. Proc. Natl. Acad. Sci. USA 107, 7305–7310 (2010).
Ma, J., Goryaynov, A., Sarma, A. & Yang, W. Self-regulated viscous channel in the nuclear pore complex. Proc. Natl. Acad. Sci. USA 109, 7326–7331 (2012).
Ma, J. et al. High-resolution three-dimensional mapping of mRNA export through the nuclear pore. Nat. Commun. 4, 2414 (2013).
Kutay, U., Izaurralde, E., Bischoff, F.R., Mattaj, I.W. & Görlich, D. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex. EMBO J. 16, 1153–1163 (1997).
Bayliss, R., Littlewood, T., Strawn, L.A., Wente, S.R. & Stewart, M. GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta. J. Biol. Chem. 277, 50597–50606 (2002).
Patel, S.S., Belmont, B.J., Sante, J.M. & Rexach, M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex. Cell 129, 83–96 (2007).
Peters, R. Translocation through the nuclear pore: Kaps pave the way. BioEssays 31, 466–477 (2009).
Ben-Efraim, I. & Gerace, L. Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import. J. Cell Biol. 152, 411–417 (2001).
Isgro, T.A. & Schulten, K. Association of nuclear pore FG-repeat domains to NTF2 import and export complexes. J. Mol. Biol. 366, 330–345 (2007).
Iwamoto, M., Asakawa, H., Hiraoka, Y. & Haraguchi, T. Nucleoporin Nup98: a gatekeeper in the eukaryotic kingdoms. Genes Cells 15, 661–669 (2010).
Katahira, J., Straesser, K., Saiwaki, T., Yoneda, Y. & Hurt, E. Complex formation between Tap and p15 affects binding to FG-repeat nucleoporins and nucleocytoplasmic shuttling. J. Biol. Chem. 277, 9242–9246 (2002).
Ghavami, A., Veenhoff, L.M., van der Giessen, E. & Onck, P.R. Probing the disordered domain of the nuclear pore complex through coarse-grained molecular dynamics simulations. Biophys. J. 107, 1393–1402 (2014).
Dange, T., Grünwald, D., Grünwald, A., Peters, R. & Kubitscheck, U. Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study. J. Cell Biol. 183, 77–86 (2008).
Wagner, R.S., Kapinos, L.E., Marshall, N.J., Stewart, M. & Lim, R.Y. Promiscuous binding of Karyopherinβ1 modulates FG nucleoporin barrier function and expedites NTF2 transport kinetics. Biophys. J. 108, 918–927 (2015).
Tetenbaum-Novatt, J., Hough, L.E., Mironska, R., McKenney, A.S. & Rout, M.P. Nucleocytoplasmic transport: a role for nonspecific competition in karyopherin-nucleoporin interactions. Mol. Cell. Proteomics 11, 31–46 (2012).
Kerr, A.R. & Schirmer, E.C. FG repeats facilitate integral protein trafficking to the inner nuclear membrane. Commun. Integr. Biol. 4, 557–559 (2011).
Yang, W., Gelles, J. & Musser, S.M. Imaging of single-molecule translocation through nuclear pore complexes. Proc. Natl. Acad. Sci. USA 101, 12887–12892 (2004).
Yang, W. & Musser, S.M. Nuclear import time and transport efficiency depend on importin beta concentration. J. Cell Biol. 174, 951–961 (2006).
Yang, W. & Musser, S.M. Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy. Methods 39, 316–328 (2006).
Sun, C., Yang, W., Tu, L.C. & Musser, S.M. Single-molecule measurements of importin alpha/cargo complex dissociation at the nuclear pore. Proc. Natl. Acad. Sci. USA 105, 8613–8618 (2008).
Mortensen, K.I., Churchman, L.S., Spudich, J.A. & Flyvbjerg, H. Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat. Methods 7, 377–381 (2010).
Quan, T., Zeng, S. & Huang, Z.L. Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging. J. Biomed. Opt. 15, 066005 (2010).
Robbins, M.S. & Hadwen, B.J. The noise performance of electron multiplying charge-coupled devices. IEEE Trans. Electron. Dev. 50, 1227–1232 (2003).
Deschout, H., Neyts, K. & Braeckmans, K. The influence of movement on the localization precision of sub-resolution particles in fluorescence microscopy. J. Biophotonics 5, 97–109 (2012).
Acknowledgements
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.).
Author information
Authors and Affiliations
Contributions
W.Y. designed experiments; J.M. and W.Y. performed experiments, established cell lines and built microscopy equipment; J.M., A.G. and W.Y. conducted data analysis; and J.M. and W.Y. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Simplified schematic of SPEED microscopy.
(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).).
Supplementary Figure 2 2D super-resolution locations in the NPC for all probes used in this paper.
All the determined locations for each probe are within a rectangular area of 240 × 160 nm around the centroid of the NPC.
Supplementary Figure 3 Graphical demonstration of the 2D-to-3D conversion process.
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).
Supplementary Figure 4 3D probability density maps of charged FG segments in the NPC.
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.
Supplementary Figure 5 The spatial distribution of FG domains in the NPC, shown in top view.
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.
Supplementary Figure 6 The competition between TRs and FG segments.
(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 information
Supplementary Text and Figures
Supplementary Figures 1–6, Supplementary Tables 1 and 2 and Supplementary Note (PDF 1658 kb)
Supplementary Data Set 1
Sequences of FG segments, Imp b1 and mutants (PDF 125 kb)
Single FG segment interacting with a single NPC
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)
Single Imp β1 imports through a single NPC
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)
Single Crm1 exports through a single NPC
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)
Three separated 3D spatial distributions of FG repeats in the native NPC
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.3174
This article is cited by
-
Physical model of the nuclear membrane permeability mechanism
Biophysical Reviews (2023)
-
Super-resolved 3D tracking of cargo transport through nuclear pore complexes
Nature Cell Biology (2022)
-
Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time
Nature Communications (2021)
-
A designer FG-Nup that reconstitutes the selective transport barrier of the nuclear pore complex
Nature Communications (2021)
-
Deconstructing transport-distribution reconstruction in the nuclear-pore complex
Nature Structural & Molecular Biology (2018)