Structure and Function of the Nuclear Pore Complex Revealed by High-Resolution Fluorescence Microscopy

  • Joseph Kelich
  • Jingjie Yu
  • Weidong YangEmail author
Part of the Nucleic Acids and Molecular Biology book series (NUCLEIC, volume 33)


Nuclear pore complexes (NPCs) are large macromolecular gateways that serve to regulate the transport of various molecules to and from the nucleus of eukaryotic cells. NPCs mediate the nuclear export of key endogenous cargoes such as mRNA, and pre-ribosomal subunits as well as allow for the nuclear import of nuclear proteins. Remarkably, other particles that are not qualified for nucleocytoplasmic transport are blocked from transport. Recently, advances in fluorescence microscopy enable live-cell and/or real-time detection of the structure and function of NPCs with high spatial and temporal resolutions. This chapter serves to summarize the advances in fluorescence microscopy techniques that have been applied to study NPC structure as well as the nucleocytoplasmic transport mechanism.


  1. Adam SA (2001) The nuclear pore complex. Genome Biol 2(9), reviews0007-1CrossRefGoogle Scholar
  2. Arhel N, Genovesio A, Kim KA, Miko S, Perret E, Olivo-Marin JC, Shorte S, Charneau P (2006) Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes. Nat Methods 3(10):817CrossRefPubMedGoogle Scholar
  3. Ben-Efraim I, Gerace L (2001) Gradient of increasing affinity of importin β for nucleoporins along the pathway of nuclear import. J Cell Biol 152(2):411–418CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bohnsack MT, Czaplinski K, Görlich D (2004) Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA 10(2):185–191CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chatel G, Desai SH, Mattheyses AL, Powers MA, Fahrenkrog B (2012) Domain topology of nucleoporin Nup98 within the nuclear pore complex. J Struct Biol 177(1):81–89CrossRefPubMedGoogle Scholar
  6. Daya S, Berns KI (2008) Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 21(4):583–593CrossRefPubMedPubMedCentralGoogle Scholar
  7. Denning DP, Patel SS, Uversky V, Fink AL, Rexach M (2003) Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded. Proc Natl Acad Sci 100(5):2450–2455CrossRefPubMedGoogle Scholar
  8. El-Tanani M, Dakir EH, Raynor B, Morgan R (2016) Mechanisms of nuclear export in cancer and resistance to chemotherapy. Cancers 8(3):35CrossRefPubMedCentralGoogle Scholar
  9. Goldberg MW, Allen TD (1995) Structural and functional organization of the nuclear envelope. Curr Opin Cell Biol 7(3):301–309CrossRefPubMedGoogle Scholar
  10. Göttfert F, Pleiner T, Heine J, Westphal V, Görlich D, Sahl SJ, Hell SW (2017) Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent. Proc Natl Acad Sci 114(9):2125–2130CrossRefPubMedGoogle Scholar
  11. Greber UF, Suomalainen M, Stidwill RP, Boucke K, Ebersold MW, Helenius A (1997) The role of the nuclear pore complex in adenovirus DNA entry. EMBO J 16(19):5998–6007CrossRefPubMedPubMedCentralGoogle Scholar
  12. Grünwald D, Singer RH (2010) In vivo imaging of labelled endogenous [bgr]-actin mRNA during nucleocytoplasmic transport. Nature 467(7315):604–607CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(2):82–87CrossRefPubMedGoogle Scholar
  14. Guo J, Amemiya S (2005) Permeability of the nuclear envelope at isolated Xenopus Oocyte nuclei studied by scanning electrochemical microscopy. Anal Chem 77(7):2147–2156CrossRefPubMedGoogle Scholar
  15. Hernandez MP, Oses C, Peña D, Criollo A, Morselli E (2016) Mutant p53 located in the cytoplasm inhibits autophagy. In: Autophagy: cancer, other pathologies, inflammation, immunity, infection, and aging. Academic Press, London, pp 189–203CrossRefGoogle Scholar
  16. Kelich JM, Yang W (2014) High-resolution imaging reveals new features of nuclear export of mRNA through the nuclear pore complexes. Int J Mol Sci 15(8):14492–14504CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kelich JM, Ma J, Dong B, Wang Q, Chin M, Magura CM, Weidong X, Yang W (2015) Super-resolution imaging of nuclear import of adeno-associated virus in live cells. Mol Ther Methods Clin Dev 2:15047CrossRefPubMedPubMedCentralGoogle Scholar
  18. Köhler A, Hurt E (2007) Exporting RNA from the nucleus to the cytoplasm. Nat Rev Mol Cell Biol 8(10):761–773CrossRefPubMedGoogle Scholar
  19. Kubitscheck U, Grünwald D, Hoekstra A, Rohleder D, Kues T, Siebrasse JP, Peters R (2005) Nuclear transport of single molecules. J Cell Biol 168(2):233–243CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kutay U, Lipowsky G, Izaurralde E, Bischoff FR, Schwarzmaier P, Hartmann E, Görlich D (1998) Identification of a tRNA-specific nuclear export receptor. Mol Cell 1(3):359–369CrossRefPubMedGoogle Scholar
  21. Le Hir H, Gatfield D, Izaurralde E, Moore MJ (2001) The exon–exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J 20(17):4987–4997CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lelek M, Di Nunzio F, Henriques R, Charneau P, Arhel N, Zimmer C (2012) Superresolution imaging of HIV in infected cells with FlAsH-PALM. Proc Natl Acad Sci 109(22):8564–8569CrossRefPubMedGoogle Scholar
  23. Leung BO, Chou KC (2011) Review of super-resolution fluorescence microscopy for biology. Appl Spectrosc 65(9):967–980CrossRefPubMedGoogle Scholar
  24. Lim RY, Huang NP, Köser J, Deng J, Lau KA, Schwarz-Herion K, Fahrenkrog B, Aebi U (2006) Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport. Proc Natl Acad Sci 103(25):9512–9517CrossRefPubMedGoogle Scholar
  25. Lim RY, Fahrenkrog B, Köser J, Schwarz-Herion K, Deng J, Aebi U (2007) Nanomechanical basis of selective gating by the nuclear pore complex. Science 318(5850):640–643CrossRefPubMedGoogle Scholar
  26. Löschberger A, Franke C, Krohne G, van de Linde S, Sauer M (2014) Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution. J Cell Sci 127(20):4351–4355CrossRefPubMedGoogle Scholar
  27. Ma J, Yang W (2010a) Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots. Proc Natl Acad Sci 107(16):7305–7310CrossRefPubMedGoogle Scholar
  28. Ma J, Yang W (2010b) Single-molecule snapshots of three-dimensional distribution of transient interactions in the nuclear pore complex. Biophys J 98(3):308aCrossRefGoogle Scholar
  29. Ma J, Goryaynov A, Sarma A, Yang W (2012) Self-regulated viscous channel in the nuclear pore complex. Proc Natl Acad Sci 109(19):7326–7331CrossRefPubMedGoogle Scholar
  30. Ma J, Liu Z, Michelotti N, Pitchiaya S, Veerapaneni R, Androsavich JR, Walter NG, Yang W (2013) High-resolution three-dimensional mapping of mRNA export through the nuclear pore. Nat Commun 4:2414CrossRefPubMedPubMedCentralGoogle Scholar
  31. Ma J, Goryaynov A, Yang W (2016) Super-resolution 3D tomography of interactions and competition in the nuclear pore complex. Nat Struct Mol Biol 23(3):239–247CrossRefPubMedPubMedCentralGoogle Scholar
  32. Ma J, Kelich JM, Junod SL, Yang W (2017) Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex. J Cell Sci 130(7):1299–1306CrossRefPubMedPubMedCentralGoogle Scholar
  33. Mattheyses AL, Kampmann M, Atkinson CE, Simon SM (2010) Fluorescence anisotropy reveals order and disorder of protein domains in the nuclear pore complex. Biophys J 99(6):1706–1717CrossRefPubMedPubMedCentralGoogle Scholar
  34. Milles S, Lemke EA (2011) Single molecule study of the intrinsically disordered FG-repeat nucleoporin 153. Biophys J 101(7):1710–1719CrossRefPubMedPubMedCentralGoogle Scholar
  35. Montpetit B, Weis K (2012) An alternative route for nuclear mRNP export by membrane budding. Science 336(6083):809–810CrossRefPubMedGoogle Scholar
  36. Mor A, Suliman S, Ben-Yishay R, Yunger S, Brody Y, Shav-Tal Y (2010) Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells. Nat Cell Biol 12(6):543–552CrossRefPubMedGoogle Scholar
  37. Nigg EA (1997) Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386(6627):779CrossRefPubMedGoogle Scholar
  38. Ojala PM, Sodeik B, Ebersold MW, Kutay U, Helenius A (2000) Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro. Mol Cell Biol 20(13):4922–4931CrossRefPubMedPubMedCentralGoogle Scholar
  39. O’Neill RE, Talon J, Palese P (1998) The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17(1):288–296CrossRefPubMedPubMedCentralGoogle Scholar
  40. Panté N, Aebi U (1993) The nuclear pore complex. J Cell Biol 122(5):977–984CrossRefPubMedGoogle Scholar
  41. Panté N, Kann M (2002) Nuclear pore complex is able to transport macromolecules with diameters of∼39 nm. Mol Biol Cell 13(2):425–434CrossRefPubMedPubMedCentralGoogle Scholar
  42. Peters R (2005) Translocation through the nuclear pore complex: selectivity and speed by reduction-of-dimensionality. Traffic 6(5):421–427CrossRefPubMedGoogle Scholar
  43. Pemberton LF, Blobel G, Rosenblum JS (1998) Transport routes through the nuclear pore complex. Curr Opin Cell Biol 10(3):392–399CrossRefPubMedGoogle Scholar
  44. Piston DW, Kremers GJ (2007) Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem Sci 32(9):407–414CrossRefPubMedGoogle Scholar
  45. Pollard VW, Malim MH (1998) The HIV-1 rev protein. Annu Rev Microbiol 52(1):491–532CrossRefPubMedGoogle Scholar
  46. Prasad BV, Rothnagel R, Jiang XI, Estes MK (1994) Three-dimensional structure of baculovirusexpressed Norwalk virus capsids. J Virol 68(8):5117–5125PubMedPubMedCentralGoogle Scholar
  47. Rexach M, Blobel G (1995) Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83(5):683–692CrossRefPubMedGoogle Scholar
  48. Ribbeck K, Görlich D (2001) Kinetic analysis of translocation through nuclear pore complexes. EMBO J 20(6):1320–1330CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rout MP, Aitchison JD, Magnasco MO, Chait BT (2003) Virtual gating and nuclear transport: the hole picture. Trends Cell Biol 13(12):622–628CrossRefPubMedGoogle Scholar
  50. Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5(6):507CrossRefPubMedPubMedCentralGoogle Scholar
  51. Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein NA, Hunter LA (1991) Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J 10(12):3941–3950PubMedPubMedCentralCrossRefGoogle Scholar
  52. Seisenberger G, Ried MU, Endress T, Büning H, Hallek M, Bräuchle C (2001) Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science 294(5548):1929–1932CrossRefPubMedGoogle Scholar
  53. Sharma M, Jamieson C, Johnson M, Molloy MP, Henderson BR (2012) Specific armadillo repeat sequences facilitate β-catenin nuclear transport in live cells via direct binding to nucleoporins Nup62, Nup153, and RanBP2/Nup358. J Biol Chem 287(2):819–831CrossRefPubMedGoogle Scholar
  54. Siebrasse JP, Kaminski T, Kubitscheck U (2012) Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy. Proc Natl Acad Sci 109(24):9426–9431CrossRefPubMedGoogle Scholar
  55. Silver PA (1991) How proteins enter the nucleus. Cell 64(3):489–497CrossRefPubMedGoogle Scholar
  56. Smith C, Lari A, Derrer CP, Ouwehand A, Rossouw A, Huisman M, Dange T, Hopman M, Joseph A, Zenklusen D, Weis K (2015a) In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p. J Cell Biol 211(6):1121–1130CrossRefPubMedPubMedCentralGoogle Scholar
  57. Smith CS, Preibisch S, Joseph A, Abrahamsson S, Rieger B, Myers E, Singer R, Grunwald D (2015b) Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking. J Cell Biol 209(4):609–619CrossRefPubMedPubMedCentralGoogle Scholar
  58. Soniat M, Chook YM (2015) Nuclear localization signals for four distinct karyopherin-β nuclear import systems. Biochem J 468(3):353–362CrossRefPubMedGoogle Scholar
  59. Stanley GJ, Fassati A, Hoogenboom BW (2017) Biomechanics of the transport barrier in the nuclear pore complex. Semin Cell Dev Biol 68:42–51CrossRefPubMedGoogle Scholar
  60. Stommel JM, Marchenko ND, Jimenez GS, Moll UM, Hope TJ, Wahl GM (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J 18(6):1660–1672CrossRefPubMedPubMedCentralGoogle Scholar
  61. Stutz F, Izaurralde E (2003) The interplay of nuclear mRNP assembly, mRNA surveillance and export. Trends Cell Biol 13(6):319–327CrossRefPubMedGoogle Scholar
  62. Sun C, Yang W, Tu LC, Musser SM (2008) Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore. Proc Natl Acad Sci 105(25):8613–8618CrossRefPubMedGoogle Scholar
  63. Sudhaharan T, Liu P, Foo YH, Bu W, Lim KB, Wohland T, Ahmed S (2009) Determination of in vivo dissociation constant, KD, of Cdc42-effector complexes in live mammalian cells using single wavelength fluorescence cross-correlation spectroscopy. J Biol Chem 284(20):13602–13609CrossRefPubMedPubMedCentralGoogle Scholar
  64. Suntharalingam M, Wente SR (2003) Peering through the pore: nuclear pore complex structure, assembly, and function. Dev Cell 4(6):775–789CrossRefPubMedGoogle Scholar
  65. Szymborska A, de Marco A, Daigle N, Cordes VC, Briggs JA, Ellenberg J (2013) Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341(6146):655–658CrossRefPubMedGoogle Scholar
  66. Takahashi Y, Itami T, Kondo M, Maeda M, Fujii R, Tomonaga S et al (1994) Electron microscopic evidence of bacilliform virus infection in kuruma shrimp (Penaeus japonicus). Fish Pathol 29(2):121–125CrossRefGoogle Scholar
  67. Terry LJ, Wente SR (2009) Flexible gates: dynamic topologies and functions for FG nucleoporins in nucleocytoplasmic transport. Eukaryot Cell 8(12):1814–1827CrossRefPubMedPubMedCentralGoogle Scholar
  68. Terry LJ, Shows EB, Wente SR (2007) Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 318(5855):1412–1416CrossRefGoogle Scholar
  69. von Appen A, Beck M (2016) Structure determination of the nuclear pore complex with three-dimensional cryo electron microscopy. J Mol Biol 428(10):2001–2010CrossRefGoogle Scholar
  70. Wang Y, Chen J, Irudayaraj J (2011) Nuclear targeting dynamics of gold nanoclusters for enhanced therapy of HER2+ breast cancer. ACS Nano 5(12):9718–9725CrossRefPubMedGoogle Scholar
  71. Weber M, Mickoleit M, Huisken J (2014) Light sheet microscopy. Methods Cell Biol 123:193–215CrossRefPubMedGoogle Scholar
  72. Weis K (2002) Nucleocytoplasmic transport: cargo trafficking across the border. Curr Opin Cell Biol 14(3):328–335CrossRefPubMedGoogle Scholar
  73. Wente SR, Rout MP (2010) The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol 2(10):a000562CrossRefPubMedPubMedCentralGoogle Scholar
  74. Wen L, Lin Y, Zheng ZH, Zhang ZL, Zhang LJ, Wang LY et al (2014) Labeling the nucleocapsid of enveloped baculovirus with quantum dots for single-virus tracking. Biomaterials 35(7):2295–2301CrossRefPubMedGoogle Scholar
  75. Whittaker GR, Kann M, Helenius A (2000) Viral entry into the nucleus. Annu Rev Cell Dev Biol 16(1):627–651CrossRefPubMedGoogle Scholar
  76. Winterflood CM, Ewers H (2014) Single-molecule localization microscopy using mCherry. ChemPhysChem 15(16):3447–3451CrossRefPubMedGoogle Scholar
  77. Yang W (2011) ‘Natively unfolded’ nucleoporins in nucleocytoplasmic transport: clustered or evenly distributed? Nucleus 2(1):10–16PubMedPubMedCentralGoogle Scholar
  78. Yang W (2013) Distinct, but not completely separate spatial transport routes in the nuclear pore complex. Nucleus 4(3):166–175CrossRefPubMedPubMedCentralGoogle Scholar
  79. Yang W, Musser SM (2006) Nuclear import time and transport efficiency depend on importin β concentration. J Cell Biol 174(7):951–961CrossRefPubMedPubMedCentralGoogle Scholar
  80. Yang Q, Rout MP, Akey CW (1998) Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications. Mol Cell 1(2):223–234CrossRefPubMedGoogle Scholar
  81. Yang W, Gelles J, Musser SM (2004) Imaging of single-molecule translocation through nuclear pore complexes. Proc Natl Acad Sci USA 101(35):12887–12892CrossRefPubMedGoogle Scholar
  82. Zincarelli C, Soltys S, Rengo G, Rabinowitz JE (2008) Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 16(6):1073–1080CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiologyTemple UniversityPhiladelphiaUSA

Personalised recommendations