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Super-resolution 3D tomography of interactions and competition in the nuclear pore complex

Nature Structural & Molecular Biology volume 23, pages 239247 (2016) | Download Citation

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.

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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

Author notes

    • Jiong Ma
    •  & Alexander Goryaynov

    Present addresses: Department of Optical Science and Engineering, Fudan University, Shanghai, China (J.M.) and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA (A.G.).

Affiliations

  1. Department of Biology, Temple University, Philadelphia, Pennsylvania, USA.

    • Jiong Ma
    • , Alexander Goryaynov
    •  & Weidong Yang

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Weidong Yang.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6, Supplementary Tables 1 and 2 and Supplementary Note

  2. 2.

    Supplementary Data Set 1

    Sequences of FG segments, Imp b1 and mutants

Videos

  1. 1.

    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).

  2. 2.

    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.

  3. 3.

    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.

  4. 4.

    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.

  5. 5.

    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.

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DOI

https://doi.org/10.1038/nsmb.3174

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