Single-Molecule Imaging to Characterize the Transport Mechanism of the Nuclear Pore Complex

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1431)

Abstract

In the eukaryotic cell, a large macromolecular channel, known as the Nuclear Pore Complex (NPC), mediates all molecular transport between the nucleus and cytoplasm. In recent years, single-molecule fluorescence (SMF) imaging has emerged as a powerful tool to study the molecular mechanism of transport through the NPC. More recently, techniques such as single-molecule localization microscopy (SMLM) have enabled the spatial and temporal distribution of cargos, transport receptors and even structural components of the NPC to be determined with nanometre accuracy. In this protocol, we describe a method to study the position and/or motion of individual molecules transiting through the NPC with high spatial and temporal precision.

Key words

Nucleus Nuclear pore complex Single-molecule tracking Super-resolution microscopy 

References

  1. 1.
    Devos D, Dokudovskaya S, Williams R, Alber F, Eswar N, Chait BT, Rout MP, Sali A (2006) Simple fold composition and modular architecture of the nuclear pore complex. Proc Natl Acad Sci U S A 103(7):2172–2177. doi:10.1073/pnas.0506345103 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Grossman E, Medalia O, Zwerger M (2012) Functional architecture of the nuclear pore complex. Annu Rev Biophys 41:557–584. doi:10.1146/annurev-biophys-050511-102328 CrossRefPubMedGoogle Scholar
  3. 3.
    Terry LJ, Shows EB, Wente SR (2007) Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 318(5855):1412–1416. doi:10.1126/science.1142204 CrossRefPubMedGoogle Scholar
  4. 4.
    Kalderon D, Roberts BL, Richardson WD, Smith AE (1984) A short amino acid sequence able to specify nuclear location. Cell 39(3 Pt 2):499–509CrossRefPubMedGoogle Scholar
  5. 5.
    Cingolani G, Petosa C, Weis K, Müller CW (1999) Structure of importin-beta bound to the IBB domain of importin-alpha. Nature 399(6733):221–229. doi:10.1038/20367 CrossRefPubMedGoogle Scholar
  6. 6.
    Ribbeck K, Görlich D (2001) Kinetic analysis of translocation through nuclear pore complexes. EMBO J 20(6):1320–1330. doi:10.1093/emboj/20.6.1320 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Izaurralde E, Kutay U, von Kobbe C, Mattaj IW, Gorlich D (1997) The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J 16(21):6535–6547. doi:10.1093/emboj/16.21.6535 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kalab P, Weis K, Heald R (2002) Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts. Science 295(5564):2452–2456. doi:10.1126/science.1068798 CrossRefPubMedGoogle Scholar
  9. 9.
    Görlich D, Panté N, Kutay U, Aebi U, Bischoff FR (1996) Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J 15(20):5584–5594PubMedPubMedCentralGoogle Scholar
  10. 10.
    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
  11. 11.
    Frey S, Richter RP, Görlich D (2006) FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314(5800):815–817. doi:10.1126/science.1132516 CrossRefPubMedGoogle Scholar
  12. 12.
    Frey S, Görlich D (2007) A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 130(3):512–523. doi:10.1016/j.cell.2007.06.024 CrossRefPubMedGoogle Scholar
  13. 13.
    Lowe AR, Tang JH, Yassif J, Graf M, Huang WY, Groves JT, Weis K, Liphardt JT (2015) Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner. eLife 4:doi:10.7554/eLife.04052CrossRefGoogle Scholar
  14. 14.
    Bestembayeva A, Kramer A, Labokha AA, Osmanović D, Liashkovich I, Orlova EV, Ford IJ, Charras G, Fassati A, Hoogenboom BW (2015) Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes. Nat Nanotechnol 10(1):60–64. doi:10.1038/nnano.2014.262 CrossRefPubMedGoogle Scholar
  15. 15.
    Adam SA, Marr RS, Gerace L (1990) Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol 111(3):807–816CrossRefPubMedGoogle Scholar
  16. 16.
    Dange T, Grünwald D, Grünwald A, Peters R, Kubitscheck U (2008) Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study. J Cell Biol 183(1):77–86. doi:10.1083/jcb.200806173 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Yang W, Musser SM (2006) Nuclear import time and transport efficiency depend on importin beta concentration. J Cell Biol 174(7):951–961. doi:10.1083/jcb.200605053 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kubitscheck U, Grünwald D, Hoekstra A, Rohleder D, Kues T, Siebrasse JP, Peters R (2005) Nuclear transport of single molecules: dwell times at the nuclear pore complex. J Cell Biol 168(2):233–243. doi:10.1083/jcb.200411005 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kahms M, Lehrich P, Hüve J, Sanetra N, Peters R (2009) Binding site distribution of nuclear transport receptors and transport complexes in single nuclear pore complexes. Traffic 10(9):1228–1242. doi:10.1111/j.1600-0854.2009.00947.x CrossRefPubMedGoogle Scholar
  20. 20.
    Tokunaga M, Imamoto N, Sakata-Sogawa K (2008) Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5(2):159–161. doi:10.1038/nmeth1171 CrossRefPubMedGoogle Scholar
  21. 21.
    Ma J, Yang W (2010) Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots. Proc Natl Acad Sci U S A 107(16):7305–7310. doi:10.1073/pnas.0908269107 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yang W (2013) Distinct, but not completely separate spatial transport routes in the nuclear pore complex. Nucleus 4(3):166–175. doi:10.4161/nucl.24874 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lowe AR, Siegel JJ, Kalab P, Siu M, Weis K, Liphardt JT (2010) Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking. Nature 467(7315):600–603. doi:10.1038/nature09285 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Sun C, Yang W, Tu LC, Musser SM (2008) Single-molecule measurements of importin alpha/cargo complex dissociation at the nuclear pore. Proc Natl Acad Sci U S A 105(25):8613–8618. doi:10.1073/pnas.0710867105 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science (NY) 313(5793):1642–1645. doi:10.1126/science.1127344 CrossRefGoogle Scholar
  26. 26.
    Rust M, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–796CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Heilemann M, van de Linde S, Mukherjee A, Sauer M (2009) Super-resolution imaging with small organic fluorophores. Angew Chem Int Ed Engl 48(37):6903–6908. doi:10.1002/anie.200902073 CrossRefPubMedGoogle Scholar
  28. 28.
    Löschberger A, van de Linde S, Dabauvalle MC, Rieger B, Heilemann M, Krohne G, Sauer M (2012) Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J Cell Sci 125(Pt 3):570–575. doi:10.1242/jcs.098822 CrossRefPubMedGoogle Scholar
  29. 29.
    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(Pt 20):4351–4355. doi:10.1242/jcs.156620 CrossRefPubMedGoogle Scholar
  30. 30.
    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. doi:10.1126/science.1240672 CrossRefPubMedGoogle Scholar
  31. 31.
    Henriques R, Lelek M, Fornasiero EF, Valtorta F, Zimmer C, Mhlanga MM (2010) QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat Methods 7(5):339–340. doi:10.1038/nmeth0510-339 CrossRefPubMedGoogle Scholar
  32. 32.
    Ovesny M, Krizek P, Borkovec J, Svindrych Z, Hagen GM (2014) ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30(16):2389–2390. doi:10.1093/bioinformatics/btu202 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Starr R, Stahlheber S, Small A (2012) Fast maximum likelihood algorithm for localization of fluorescent molecules. Opt Lett 37(3):413–415CrossRefPubMedGoogle Scholar
  34. 34.
    Crocker J, Grier D (1996) Methods of digital video microscopy for colloidal studies. J Colloid Interface Sci 179(1):298–310CrossRefGoogle Scholar
  35. 35.
    Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL, Danuser G (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5(8):695–702. doi:10.1038/nmeth.1237 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Thompson R, Larson D, Webb W (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Structural & Molecular BiologyUniversity College LondonLondonUK
  2. 2.Department of Biological Sciences, Birkbeck CollegeUniversity of LondonLondonUK
  3. 3.London Centre for NanotechnologyLondonUK

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