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

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Nuclear-Cytoplasmic Transport

Part of the book series: Nucleic Acids and Molecular Biology ((NUCLEIC,volume 33))

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Abstract

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.

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References

  • Adam SA (2001) The nuclear pore complex. Genome Biol 2(9), reviews0007-1

    Article  Google Scholar 

  • 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):817

    Article  CAS  PubMed  Google Scholar 

  • 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):411–418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):185–191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):81–89

    Article  CAS  PubMed  Google Scholar 

  • Daya S, Berns KI (2008) Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 21(4):583–593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):2450–2455

    Article  CAS  PubMed  Google Scholar 

  • El-Tanani M, Dakir EH, Raynor B, Morgan R (2016) Mechanisms of nuclear export in cancer and resistance to chemotherapy. Cancers 8(3):35

    Article  CAS  PubMed Central  Google Scholar 

  • Goldberg MW, Allen TD (1995) Structural and functional organization of the nuclear envelope. Curr Opin Cell Biol 7(3):301–309

    Article  CAS  PubMed  Google Scholar 

  • 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):2125–2130

    Article  CAS  PubMed  Google Scholar 

  • 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):5998–6007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grünwald D, Singer RH (2010) In vivo imaging of labelled endogenous [bgr]-actin mRNA during nucleocytoplasmic transport. Nature 467(7315):604–607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(2):82–87

    Article  CAS  PubMed  Google Scholar 

  • Guo J, Amemiya S (2005) Permeability of the nuclear envelope at isolated Xenopus Oocyte nuclei studied by scanning electrochemical microscopy. Anal Chem 77(7):2147–2156

    Article  CAS  PubMed  Google Scholar 

  • 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 189–203

    Chapter  Google Scholar 

  • 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):14492–14504

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 2:15047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Köhler A, Hurt E (2007) Exporting RNA from the nucleus to the cytoplasm. Nat Rev Mol Cell Biol 8(10):761–773

    Article  CAS  PubMed  Google Scholar 

  • 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):233–243

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):359–369

    Article  CAS  PubMed  Google Scholar 

  • 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):4987–4997

    Article  PubMed  PubMed Central  Google Scholar 

  • 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):8564–8569

    Article  PubMed  Google Scholar 

  • Leung BO, Chou KC (2011) Review of super-resolution fluorescence microscopy for biology. Appl Spectrosc 65(9):967–980

    Article  CAS  PubMed  Google Scholar 

  • 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):9512–9517

    Article  CAS  PubMed  Google Scholar 

  • 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):640–643

    Article  CAS  PubMed  Google Scholar 

  • 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):4351–4355

    Article  CAS  PubMed  Google Scholar 

  • 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):7305–7310

    Article  PubMed  Google Scholar 

  • Ma J, Yang W (2010b) Single-molecule snapshots of three-dimensional distribution of transient interactions in the nuclear pore complex. Biophys J 98(3):308a

    Article  Google Scholar 

  • Ma J, Goryaynov A, Sarma A, Yang W (2012) Self-regulated viscous channel in the nuclear pore complex. Proc Natl Acad Sci 109(19):7326–7331

    Article  PubMed  Google Scholar 

  • 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 4:2414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):239–247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):1299–1306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):1706–1717

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Milles S, Lemke EA (2011) Single molecule study of the intrinsically disordered FG-repeat nucleoporin 153. Biophys J 101(7):1710–1719

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Montpetit B, Weis K (2012) An alternative route for nuclear mRNP export by membrane budding. Science 336(6083):809–810

    Article  CAS  PubMed  Google Scholar 

  • 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):543–552

    Article  CAS  PubMed  Google Scholar 

  • Nigg EA (1997) Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386(6627):779

    Article  CAS  PubMed  Google Scholar 

  • 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):4922–4931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):288–296

    Article  PubMed  PubMed Central  Google Scholar 

  • Panté N, Aebi U (1993) The nuclear pore complex. J Cell Biol 122(5):977–984

    Article  PubMed  Google Scholar 

  • Panté N, Kann M (2002) Nuclear pore complex is able to transport macromolecules with diameters of∼39 nm. Mol Biol Cell 13(2):425–434

    Article  PubMed  PubMed Central  Google Scholar 

  • Peters R (2005) Translocation through the nuclear pore complex: selectivity and speed by reduction-of-dimensionality. Traffic 6(5):421–427

    Article  CAS  PubMed  Google Scholar 

  • Pemberton LF, Blobel G, Rosenblum JS (1998) Transport routes through the nuclear pore complex. Curr Opin Cell Biol 10(3):392–399

    Article  CAS  PubMed  Google Scholar 

  • Piston DW, Kremers GJ (2007) Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem Sci 32(9):407–414

    Article  CAS  PubMed  Google Scholar 

  • Pollard VW, Malim MH (1998) The HIV-1 rev protein. Annu Rev Microbiol 52(1):491–532

    Article  CAS  PubMed  Google Scholar 

  • Prasad BV, Rothnagel R, Jiang XI, Estes MK (1994) Three-dimensional structure of baculovirusexpressed Norwalk virus capsids. J Virol 68(8):5117–5125

    PubMed  PubMed Central  CAS  Google Scholar 

  • Rexach M, Blobel G (1995) Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83(5):683–692

    Article  CAS  PubMed  Google Scholar 

  • Ribbeck K, Görlich D (2001) Kinetic analysis of translocation through nuclear pore complexes. EMBO J 20(6):1320–1330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rout MP, Aitchison JD, Magnasco MO, Chait BT (2003) Virtual gating and nuclear transport: the hole picture. Trends Cell Biol 13(12):622–628

    Article  CAS  PubMed  Google Scholar 

  • Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5(6):507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):3941–3950

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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):1929–1932

    Article  CAS  PubMed  Google Scholar 

  • 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):819–831

    Article  CAS  PubMed  Google Scholar 

  • 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):9426–9431

    Article  PubMed  Google Scholar 

  • Silver PA (1991) How proteins enter the nucleus. Cell 64(3):489–497

    Article  CAS  PubMed  Google Scholar 

  • 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):1121–1130

    Article  PubMed  PubMed Central  Google Scholar 

  • 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):609–619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Soniat M, Chook YM (2015) Nuclear localization signals for four distinct karyopherin-β nuclear import systems. Biochem J 468(3):353–362

    Article  CAS  PubMed  Google Scholar 

  • Stanley GJ, Fassati A, Hoogenboom BW (2017) Biomechanics of the transport barrier in the nuclear pore complex. Semin Cell Dev Biol 68:42–51

    Article  CAS  PubMed  Google Scholar 

  • 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):1660–1672

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stutz F, Izaurralde E (2003) The interplay of nuclear mRNP assembly, mRNA surveillance and export. Trends Cell Biol 13(6):319–327

    Article  CAS  PubMed  Google Scholar 

  • 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):8613–8618

    Article  PubMed  Google Scholar 

  • 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):13602–13609

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suntharalingam M, Wente SR (2003) Peering through the pore: nuclear pore complex structure, assembly, and function. Dev Cell 4(6):775–789

    Article  CAS  PubMed  Google Scholar 

  • 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):655–658

    Article  CAS  PubMed  Google Scholar 

  • 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):121–125

    Article  Google Scholar 

  • Terry LJ, Wente SR (2009) Flexible gates: dynamic topologies and functions for FG nucleoporins in nucleocytoplasmic transport. Eukaryot Cell 8(12):1814–1827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Terry LJ, Shows EB, Wente SR (2007) Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 318(5855):1412–1416

    Article  CAS  Google Scholar 

  • von Appen A, Beck M (2016) Structure determination of the nuclear pore complex with three-dimensional cryo electron microscopy. J Mol Biol 428(10):2001–2010

    Article  CAS  Google Scholar 

  • Wang Y, Chen J, Irudayaraj J (2011) Nuclear targeting dynamics of gold nanoclusters for enhanced therapy of HER2+ breast cancer. ACS Nano 5(12):9718–9725

    Article  CAS  PubMed  Google Scholar 

  • Weber M, Mickoleit M, Huisken J (2014) Light sheet microscopy. Methods Cell Biol 123:193–215

    Article  PubMed  Google Scholar 

  • Weis K (2002) Nucleocytoplasmic transport: cargo trafficking across the border. Curr Opin Cell Biol 14(3):328–335

    Article  CAS  PubMed  Google Scholar 

  • Wente SR, Rout MP (2010) The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol 2(10):a000562

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):2295–2301

    Article  CAS  PubMed  Google Scholar 

  • Whittaker GR, Kann M, Helenius A (2000) Viral entry into the nucleus. Annu Rev Cell Dev Biol 16(1):627–651

    Article  CAS  PubMed  Google Scholar 

  • Winterflood CM, Ewers H (2014) Single-molecule localization microscopy using mCherry. ChemPhysChem 15(16):3447–3451

    Article  CAS  PubMed  Google Scholar 

  • Yang W (2011) ‘Natively unfolded’ nucleoporins in nucleocytoplasmic transport: clustered or evenly distributed? Nucleus 2(1):10–16

    PubMed  PubMed Central  Google Scholar 

  • Yang W (2013) Distinct, but not completely separate spatial transport routes in the nuclear pore complex. Nucleus 4(3):166–175

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang W, Musser SM (2006) Nuclear import time and transport efficiency depend on importin β concentration. J Cell Biol 174(7):951–961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):223–234

    Article  CAS  PubMed  Google Scholar 

  • Yang W, Gelles J, Musser SM (2004) Imaging of single-molecule translocation through nuclear pore complexes. Proc Natl Acad Sci USA 101(35):12887–12892

    Article  CAS  PubMed  Google Scholar 

  • 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):1073–1080

    Article  CAS  PubMed  Google Scholar 

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Kelich, J., Yu, J., Yang, W. (2018). Structure and Function of the Nuclear Pore Complex Revealed by High-Resolution Fluorescence Microscopy. In: Yang, W. (eds) Nuclear-Cytoplasmic Transport. Nucleic Acids and Molecular Biology, vol 33. Springer, Cham. https://doi.org/10.1007/978-3-319-77309-4_11

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