Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy

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 Xenopuslaevis 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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Observing native NPCs by HS-AFM.
Figure 2: HS-AFM resolves dynamic FG Nup behaviour inside an individual NPC.
Figure 3: Spatiotemporal averaging of FG Nup behaviour.
Figure 4: Entropic exclusion works in both space and time.

Similar content being viewed by others

References

  1. Grünwald, D., Singer, R. H. & Rout, M. Nuclear export dynamics of RNA-protein complexes. Nature 475, 333–341 (2011).

    Article  Google Scholar 

  2. Popken, P., Ghavami, A., Onck, P. R., Poolman, B. & Veenhoff, L. M. Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex. Mol. Biol. Cell 26, 1386–1394 (2015).

    Article  CAS  Google Scholar 

  3. 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).

    Article  CAS  Google Scholar 

  4. Stoffler, D. et al. Cryo-electron tomography provides novel insights into nuclear pore architecture Implications for nucleocytoplasmic transport. J. Mol. Biol. 328, 119–130 (2003).

    Article  CAS  Google Scholar 

  5. Beck, M. et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306, 1387–1390 (2004).

    Article  CAS  Google Scholar 

  6. Eibauer, M. et al. Structure and gating of the nuclear pore complex. Nature Commun. 6, 7532 (2015).

    Article  CAS  Google Scholar 

  7. Rout, M. P. et al. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 (2000).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Lim, R. Y. H. et al. Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport. Proc. Natl Acad. Sci. USA 103, 9512–9517 (2006).

    Article  CAS  Google Scholar 

  10. Lim, R. Y. H. et al. Nanomechanical basis of selective gating by the nuclear pore complex. Science 318, 640–643 (2007).

    Article  CAS  Google Scholar 

  11. Frey, S. & Görlich, D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 130, 512–523 (2007).

    Article  CAS  Google Scholar 

  12. Hülsmann, B. B., Labokha, A. A. & Görlich, D. The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model. Cell 150, 738–751 (2012).

    Article  Google Scholar 

  13. Akey, C. W. Visualization of transport-related configurations of the nuclear-pore transporter. Biophys. J. 58, 341–355 (1990).

    Article  CAS  Google Scholar 

  14. 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).

    Article  CAS  Google Scholar 

  15. Fahrenkrog, B. et al. Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex. J. Struct. Biol. 140, 254–267 (2002).

    Article  CAS  Google Scholar 

  16. Stoffler, D., Goldie, K. N., Feja, B. & Aebi, U. Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy. J. Mol. Biol. 287, 741–752 (1999).

    Article  CAS  Google Scholar 

  17. Bestembayeva, A. et al. Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes. Nature Nanotech. 10, 60–64 (2015).

    Article  CAS  Google Scholar 

  18. Kramer, A., Liashkovich, I., Ludwig, Y. & Shahin, V. Atomic force microscopy visualises a hydrophobic meshwork in the central channel of the nuclear pore. Pflugers Arch. 456, 155–162 (2008).

    Article  CAS  Google Scholar 

  19. Cardarelli, F., Lanzano, L. & Gratton, E. Capturing directed molecular motion in the nuclear pore complex of live cells. Proc. Natl Acad. Sci. USA 109, 9863–9868 (2012).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Ma, J., Goryaynov, A. & Yang, W. Super-resolution 3D tomography of interactions and competition in the nuclear pore complex. Nature Struct. Mol. Biol. 23, 239–247 (2016).

    Article  CAS  Google Scholar 

  22. Ando, T., Uchihashi, T. & Fukuma, T. High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes. Prog. Surf. Sci. 83, 337–437 (2008).

    Article  CAS  Google Scholar 

  23. Uchihashi, T., Kodera, N. & Ando, T. Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nature Protoc. 7, 1193–1206 (2012).

    Article  CAS  Google Scholar 

  24. Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010).

    Article  CAS  Google Scholar 

  25. Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force microscopy reveals rotary catalysis of rotorless F-1-ATPase. Science 333, 755–758 (2011).

    Article  CAS  Google Scholar 

  26. Miyagi, A. et al. Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy. ChemPhysChem 9, 1859–1866 (2008).

    Article  CAS  Google Scholar 

  27. Chatel, G., Desai, S. H., Mattheyses, A. L., Powers, M. A. & Fahrenkrog, B. Domain topology of nucleoporin Nup98 within the nuclear pore complex. J. Struct. Biol. 177, 81–89 (2012).

    Article  CAS  Google Scholar 

  28. Kapinos, L. E., Schoch, R. L., Wagner, R. S., Schleicher, K. D. & Lim, R. Y. H. Karyopherin-centric control of nuclear pores based on molecular occupancy and kinetic analysis of multivalent binding with FG nucleoporins. Biophys. J. 106, 1751–1762 (2014).

    Article  CAS  Google Scholar 

  29. Vesenka, J., Manne, S., Giberson, R., Marsh, T. & Henderson, E. Colloidal gold particles as an incompressible atomic-force microscope imaging standard for assessing the compressibility of biomolecules. Biophys. J. 65, 992–997 (1993).

    Article  CAS  Google Scholar 

  30. Chattopadhyay, K., Elson, E. L. & Frieden, C. The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods. Proc. Natl Acad. Sci. USA 102, 2385–2389 (2005).

    Article  CAS  Google Scholar 

  31. Windisch, B., Bray, D. & Duke, T. Balls and chains - A mesoscopic approach to tethered protein domains. Biophys. J. 91, 2383–2392 (2006).

    Article  CAS  Google Scholar 

  32. Kusumi, A., Sako, Y. & Yamamoto, M. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys. J. 65, 2021–2040 (1993).

    Article  CAS  Google Scholar 

  33. Schmidt, H. B. & Görlich, D. Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity. eLife 4, e04251 (2015).

    Article  Google Scholar 

  34. Osmanovic, D. et al. Bistable collective behavior of polymers tethered in a nanopore. Phys. Rev. E 85, 061917 (2012).

    Article  Google Scholar 

  35. Grünwald, D. & Singer, R. H. In vivo imaging of labelled endogenous b-actin mRNA during nucleocytoplasmic transport. Nature 467, 604–609 (2010).

    Article  Google Scholar 

  36. Tagliazucchi, M., Peleg, O., Kroger, M., Rabin, Y. & Szleifer, I. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex. Proc. Natl Acad. Sci. USA 110, 3363–3368 (2013).

    Article  CAS  Google Scholar 

  37. Ando, D. et al. Nuclear pore complex protein sequences determine overall copolymer brush structure and function. Biophys. J. 106, 1997–2007 (2014).

    Article  CAS  Google Scholar 

  38. 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).

    Article  CAS  Google Scholar 

  39. Mincer, J. S. & Simon, S. M. Simulations of nuclear pore transport yield mechanistic insights and quantitative predictions. Proc. Natl Acad. Sci. USA 108, E351–E358 (2011).

    Article  CAS  Google Scholar 

  40. Gamini, R., Han, W., Stone, J. E. & Schulten, K. Assembly of Nsp1 nucleoporins provides insight into nuclear pore complex gating. PLoS Comp. Biol. 10, e1003488 (2014).

    Article  Google Scholar 

  41. Peyro, M., Soheilypour, M., Ghavami, A. & Mofrad, M. R. K. Nucleoporin's like charge regions are major regulators of FG coverage and dynamics inside the nuclear pore complex. PLoS ONE 10, e0143745 (2015).

    Article  Google Scholar 

  42. Ando, T. High-speed atomic force microscopy. Microscopy 62, 81–93 (2013).

    Article  CAS  Google Scholar 

  43. Schmidt, H. B. & Görlich, D. Transport selectivity of nuclear pores, phase separation, and membraneless organelles. Trends Biochem. Sci. 41, 46–61 (2016).

    Article  CAS  Google Scholar 

  44. Hough, L. E. et al. The molecular mechanism of nuclear transport revealed by atomic-scale measurements. eLife 4, e10027 (2015).

    Article  Google Scholar 

  45. Milles, S. et al. Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors. Cell 163, 734–745 (2015).

    Article  CAS  Google Scholar 

  46. Hoh, J. H. Functional protein domains from the thermally driven motion of polypeptide chains A proposal. Proteins 32, 223–228 (1998).

    Article  CAS  Google Scholar 

  47. Fuxreiter, M. et al. Disordered proteinaceous machines. Chem. Rev. 114, 6806–6843 (2014).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Roderick Y. H. Lim.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 1042 kb)

Supplementary information

Supplementary Movie 1 (AVI 248 kb)

Supplementary information

Supplementary Movie 2 (AVI 395 kb)

Supplementary information

Supplementary Movie 3 (AVI 88 kb)

Supplementary information

Supplementary Movie 4 (AVI 183 kb)

Supplementary information

Supplementary Movie 5 (AVI 1383 kb)

Supplementary information

Supplementary Movie 6 (AVI 792 kb)

Supplementary information

Supplementary Movie 7 (AVI 78 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2016.62

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing