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Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes

Abstract

The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than 5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC1. Although the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized2,3,4,5, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins5,6, and is therefore not well understood. Here, we show that stiffness topography7 with sharp atomic force microscopy tips can generate nanoscale cross-sections of the NPC. The cross-sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy2,3,4,5. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel.

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Figure 1: NPCs imaged and probed by AFM.
Figure 2: Structure and nanomechanical properties of NPCs.
Figure 3: Nups modelled as interacting polymers in a cylindrical geometry.

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Acknowledgements

The authors thank H. Oberleithner for the use of laboratory facilities, P. Vergani for assistance with the preparation of X. laevis oocytes, D. Görlich for the expression plasmids for importin β and Ran-mix, S. Frey for Rch1-IBB-MBP-GFP protein, R.P. Richter for providing the data presented in Supplementary Fig. 8, J. Grech for the electron microscopy image in Fig. 1a, M. Goldberg for the protocol for removal of the nuclear basket from the NPCs, J. Bailey and A.H. Harker for support in initializing the modelling work, R. Thorogate for assistance with the confocal microscopy measurements, C. Leung for assistance in formatting the figures and T. Duke (deceased) and G. Aeppli for proofreading the manuscript. This work was partially funded by the European Molecular Biology Organization (ALTF 757-2008 to A.K.), the Kazakh Ministry of Education and Science (A.B.), the Sackler Foundation (D.O.), the UK Biotechnology and Biological Sciences Research Council (BB/G011729/1 to B.W.H.) and the Wellcome Trust (083810/Z/07/Z to A.F.). G.C. is a Royal Society University Research Fellow.

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A.B., A.K., A.L., I.L., G.C., A.F. and B.W.H. designed the experiments. A.B., A.K., A.L. and I.L. performed the experiments. A.B., A.K. and B.W.H., with support from E.V.O., performed the data analysis. D.O., I.J.F. and B.W.H. designed and performed the polymer modelling. A.F. and B.W.H. wrote the manuscript. All authors read and commented on the manuscript.

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Correspondence to Ariberto Fassati or Bart W. Hoogenboom.

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Bestembayeva, A., Kramer, A., Labokha, 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). https://doi.org/10.1038/nnano.2014.262

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