Abstract
Nuclear pore complexes (NPCs) are biological nanomachines that mediate the bidirectional traffic of macromolecules between the cytoplasm and nucleus in eukaryotic cells. This process involves numerous intrinsically disordered, barrier-forming proteins known as phenylalanine-glycine nucleoporins (FG Nups) that are tethered inside each pore. The selective barrier mechanism has so far remained unresolved because the FG Nups have eluded direct structural analysis within NPCs. Here, high-speed atomic force microscopy is used to visualize the nanoscopic spatiotemporal dynamics of FG Nups inside Xenopus laevis oocyte NPCs at timescales of ∼100 ms. Our results show that the cytoplasmic orifice is circumscribed by highly flexible, dynamically fluctuating FG Nups that rapidly elongate and retract, consistent with the diffusive motion of tethered polypeptide chains. On this basis, intermingling FG Nups exhibit transient entanglements in the central channel, but do not cohere into a tightly crosslinked meshwork. Therefore, the basic functional form of the NPC barrier is comprised of highly dynamic FG Nups that manifest as a central plug or transporter when averaged in space and time.
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Acknowledgements
We are grateful to D. Mathys, M. Dueggelin and M. Dürrenberger for EM and FIB; T. Ando, N. Kodera and T. Uchihashi for HS-AFM support; R. Strittmatter and S. Saner for machining and electronics, respectively; N. Ehrenfeucter and H. Stahlberg for advice on image processing; and Ch. Gerber for stimulating discussions. Y.S. is supported by a PhD Fellowship from the Swiss Nanoscience Institute. R.Y.H.L. is funded by the Swiss Nanoscience Institute and the Biozentrum at the University of Basel, as well as the Swiss National Science Foundation as part of the NCCR in Molecular Systems Engineering.
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Y.S. and R.Y.H.L. conceived the study and designed the experiments. Y.S. performed the HS-AFM experiments. A.M. wrote customized software for data analysis and image processing. L.E.K. extracted Xenopus laevis oocytes, contributed materials and conducted dynamic light scattering experiments. R.Y.H.L. wrote the paper with input from Y.S. and A.M.. All authors analysed data, discussed the results and commented on the manuscript.
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Sakiyama, Y., Mazur, A., Kapinos, L. et al. Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy. Nature Nanotech 11, 719–723 (2016). https://doi.org/10.1038/nnano.2016.62
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DOI: https://doi.org/10.1038/nnano.2016.62
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