Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy
Source abstract
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.
Source: europepmc · Quality: candidate unverified · Source record ↗
Research context
OpenAlex topics: Nuclear Structure and Function · RNA Research and Splicing · Force Microscopy Techniques and Applications
SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.
References (47)
Show source-provided references
1. Grünwald, D., Singer, R. H. & Rout, M. Nuclear export dynamics of RNA-protein complexes. Nature 475, 333–341 (2011).10.1038/nature10318 ↗
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).10.1091/mbc.E14-07-1175 ↗
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).10.1074/mcp.M000035-MCP201 ↗
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).10.1016/S0022-2836(03)00266-3 ↗
5. Beck, M. et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306, 1387–1390 (2004).10.1126/science.1104808 ↗
6. Eibauer, M. et al. Structure and gating of the nuclear pore complex. Nature Commun. 6, 7532 (2015).10.1038/ncomms8532 ↗
7. Rout, M. P. et al. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 (2000).10.1083/jcb.148.4.635 ↗
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).10.1016/j.tcb.2003.10.007 ↗
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).10.1073/pnas.0603521103 ↗
10. Lim, R. Y. H. et al. Nanomechanical basis of selective gating by the nuclear pore complex. Science 318, 640–643 (2007).10.1126/science.1145980 ↗
11. Frey, S. & Görlich, D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 130, 512–523 (2007).10.1016/j.cell.2007.06.024 ↗
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).10.1016/j.cell.2012.07.019 ↗
13. Akey, C. W. Visualization of transport-related configurations of the nuclear-pore transporter. Biophys. J. 58, 341–355 (1990).10.1016/S0006-3495(90)82381-X ↗
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).10.1083/jcb.200806173 ↗
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).10.1016/S1047-8477(02)00524-5 ↗
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).10.1006/jmbi.1999.2637 ↗
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).10.1038/nnano.2014.262 ↗
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).10.1007/s00424-007-0396-y ↗
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).10.1073/pnas.1200486109 ↗
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).10.1073/pnas.1201724109 ↗
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).10.1038/nsmb.3174 ↗
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).10.1016/j.progsurf.2008.09.001 ↗
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).10.1038/nprot.2012.047 ↗
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).10.1038/nature09450 ↗
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).10.1126/science.1205510 ↗
26. Miyagi, A. et al. Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy. ChemPhysChem 9, 1859–1866 (2008).10.1002/cphc.200800210 ↗
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).10.1016/j.jsb.2011.11.004 ↗
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).10.1016/j.bpj.2014.02.021 ↗
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).10.1016/S0006-3495(93)81171-8 ↗
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).10.1073/pnas.0500127102 ↗
31. Windisch, B., Bray, D. & Duke, T. Balls and chains - A mesoscopic approach to tethered protein domains. Biophys. J. 91, 2383–2392 (2006).10.1529/biophysj.105.078543 ↗
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).10.1016/S0006-3495(93)81253-0 ↗
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).10.7554/eLife.04251 ↗
34. Osmanovic, D. et al. Bistable collective behavior of polymers tethered in a nanopore. Phys. Rev. E 85, 061917 (2012).10.1103/PhysRevE.85.061917 ↗
35. Grünwald, D. & Singer, R. H. In vivo imaging of labelled endogenous b-actin mRNA during nucleocytoplasmic transport. Nature 467, 604–609 (2010).10.1038/nature09438 ↗
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).10.1073/pnas.1212909110 ↗
37. Ando, D. et al. Nuclear pore complex protein sequences determine overall copolymer brush structure and function. Biophys. J. 106, 1997–2007 (2014).10.1016/j.bpj.2014.03.021 ↗
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).10.1016/j.bpj.2014.07.060 ↗
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).10.1073/pnas.1104521108 ↗
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).10.1371/journal.pcbi.1003488 ↗
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).10.1371/journal.pone.0143745 ↗
42. Ando, T. High-speed atomic force microscopy. Microscopy 62, 81–93 (2013).10.1093/jmicro/dfs093 ↗
43. Schmidt, H. B. & Görlich, D. Transport selectivity of nuclear pores, phase separation, and membraneless organelles. Trends Biochem. Sci. 41, 46–61 (2016).10.1016/j.tibs.2015.11.001 ↗
44. Hough, L. E. et al. The molecular mechanism of nuclear transport revealed by atomic-scale measurements. eLife 4, e10027 (2015).10.7554/eLife.10027 ↗
45. Milles, S. et al. Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors. Cell 163, 734–745 (2015).10.1016/j.cell.2015.09.047 ↗
46. Hoh, J. H. Functional protein domains from the thermally driven motion of polypeptide chains A proposal. Proteins 32, 223–228 (1998).10.1002/(SICI)1097-0134(19980801)32:2<223::AID-PROT8>3.0.CO;2-L ↗
47. Fuxreiter, M. et al. Disordered proteinaceous machines. Chem. Rev. 114, 6806–6843 (2014).10.1021/cr4007329 ↗