Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking (original) (raw)
- Letter
- Published: 01 September 2010
- Jake J. Siegel2,4 na1,
- Petr Kalab5 nAff8,
- Merek Siu4 nAff8,
- Karsten Weis2,5 &
- …
- Jan T. Liphardt1,2,3,4,6
Nature volume 467, pages 600–603 (2010)Cite this article
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Abstract
The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate1. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality1, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.
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References
- Peters, R. Translocation through the nuclear pore: Kaps pave the way. Bioessays 31, 466–477 (2009)
Article CAS Google Scholar - Stewart, M. Molecular mechanism of the nuclear protein import cycle. Nature Rev. Mol. Cell Biol. 8, 195–208 (2007)
Article CAS Google Scholar - Weis, K. The nuclear pore complex: oily spaghetti or gummy bear? Cell 130, 405–407 (2007)
Article CAS Google Scholar - Frey, S. & Gorlich, D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 130, 512–523 (2007)
Article CAS Google Scholar - Frey, S., Richter, R. P. & Gorlich, D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314, 815–817 (2006)
Article ADS CAS Google Scholar - Ribbeck, K. & Gorlich, D. Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 20, 1320–1330 (2001)
Article CAS Google Scholar - 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)
Article CAS Google Scholar - Peters, R. Translocation through the nuclear pore complex: selectivity and speed by reduction-of-dimensionality. Traffic 6, 421–427 (2005)
Article CAS Google Scholar - Lim, R. Y. et al. Nanomechanical basis of selective gating by the nuclear pore complex. Science 318, 640–643 (2007)
Article ADS CAS Google Scholar - Lim, R. Y. et al. Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport. Proc. Natl Acad. Sci. USA 103, 9512–9517 (2006)
Article ADS CAS Google Scholar - Lim, R. Y., Koser, J., Huang, N. P., Schwarz-Herion, K. & Aebi, U. Nanomechanical interactions of phenylalanine-glycine nucleoporins studied by single molecule force-volume spectroscopy. J. Struct. Biol. 159, 277–289 (2007)
Article CAS Google Scholar - Patel, S. S., Belmont, B. J., Sante, J. M. & Rexach, M. F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex. Cell 129, 83–96 (2007)
Article CAS Google Scholar - Dange, T., Grunwald, D., Grunwald, 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)
Article CAS Google Scholar - Kubitscheck, U. et al. Nuclear transport of single molecules: dwell times at the nuclear pore complex. J. Cell Biol. 168, 233–243 (2005)
Article Google Scholar - Yang, W., Gelles, J. & Musser, S. M. Imaging of single-molecule translocation through nuclear pore complexes. Proc. Natl Acad. Sci. USA 101, 12887–12892 (2004)
Article ADS CAS Google Scholar - Lacoste, T. D. et al. Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc. Natl Acad. Sci. USA 97, 9461–9466 (2000)
Article ADS CAS Google Scholar - Huber, J., Dickmanns, A. & Luhrmann, R. The importin-beta binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro . J. Cell Biol. 156, 467–479 (2002)
Article CAS Google Scholar - Mitrousis, G., Olia, A. S., Walker-Kopp, N. & Cingolani, G. Molecular basis for the recognition of snurportin 1 by importin beta. J. Biol. Chem. 283, 7877–7884 (2008)
Article CAS Google Scholar - Arhel, N. et al. Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes. Nature Methods 3, 817–824 (2006)
Article CAS Google Scholar - Bukrinsky, M. I. et al. Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc. Natl Acad. Sci. USA 89, 6580–6584 (1992)
Article ADS CAS Google Scholar - Pante, N. & Kann, M. Nuclear pore complex is able to transport macromolecules with diameters of ∼39 nm. Mol. Biol. Cell 13, 425–434 (2002)
Article CAS Google Scholar - Rabe, B., Vlachou, A., Pante, N., Helenius, A. & Kann, M. Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc. Natl Acad. Sci. USA 100, 9849–9854 (2003)
Article ADS CAS Google Scholar - Adam, S. A., Marr, R. S. & Gerace, L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111, 807–816 (1990)
Article CAS Google Scholar - Beck, M., Lucic, V., Forster, F., Baumeister, W. & Medalia, O. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature 449, 611–615 (2007)
Article ADS CAS Google Scholar - Pyhtila, B. & Rexach, M. A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex. J. Biol. Chem. 278, 42699–42709 (2003)
Article CAS Google Scholar - Bickel, T. & Bruinsma, R. The nuclear pore complex mystery and anomalous diffusion in reversible gels. Biophys. J. 83, 3079–3087 (2002)
Article CAS Google Scholar - Gorlich, D., Pante, N., Kutay, U., Aebi, U. & Bischoff, F. R. Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J. 15, 5584–5594 (1996)
Article CAS Google Scholar - Moore, M. S. & Blobel, G. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365, 661–663 (1993)
Article ADS CAS Google Scholar
Acknowledgements
We thank H. Agarwal for help with initial experiments; H. Aaron, A. Fischer and B. Cohen for use of facilities and discussions; the Bustamante, Chu and Krantz laboratories for use of equipment; and C. Bustamante, M. Welch and D. Grünwald for discussions and comments on the manuscript. This work was partially funded by the NIH (GM058065 to K.W. and GM77856 to J.T.L.) and the NCI (U54CA143836 to J.T.L.).
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Author notes
- Petr Kalab & Merek Siu
Present address: Present addresses: Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA (P.K.); Illumina Inc., Hayward, California 94545, USA (M.S.)., - Alan R. Lowe and Jake J. Siegel: These authors contributed equally to this work.
Authors and Affiliations
- Department of Physics, University of California, Berkeley, 94720, California, USA
Alan R. Lowe & Jan T. Liphardt - QB3, University of California, Berkeley, 94720, California, USA
Alan R. Lowe, Jake J. Siegel, Karsten Weis & Jan T. Liphardt - Bay Area Physical Sciences–Oncology Center, University of California, Berkeley, 94720, California, USA
Alan R. Lowe & Jan T. Liphardt - Biophysics Graduate Group, University of California, Berkeley, 94720, California, USA
Jake J. Siegel, Merek Siu & Jan T. Liphardt - Department of Molecular and Cellular Biology, University of California, Berkeley, 94720, California, USA
Petr Kalab & Karsten Weis - Physical Biosciences Division, Lawrence Berkeley National Laboratory, California 94720, USA,
Alan R. Lowe & Jan T. Liphardt
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- Alan R. Lowe
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Contributions
A.R.L., J.J.S., P.K., M.S., K.W. and J.T.L. designed the experiments. A.R.L., J.J.S. and P.K. prepared materials. A.R.L. and J.J.S. performed the QD optimization and functionalization, the single-molecule experiments, and the data analysis. A.R.L., J.J.S., K.W. and J.T.L. wrote the manuscript.
Corresponding author
Correspondence toKarsten Weis.
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Supplementary information
Supplementary Information
This file contains Supplementary Information 1-8, Supplementary Figures 1-13 with legends, Supplementary Tables 1-3 and additional references. (PDF 4589 kb)
Supplementary Movie 1
A typical movie showing imported QDs in a single nucleus after 20 minutes of import. 2 QDs are seen to diffuse inside the confined volume of the cell nucleus. The fitted outline of the nuclear envelope taken from a brightfield image taken immediately after this movie is overlaid in green. (AVI 25084 kb)
Supplementary Movie 2
An example of single QD cargo successfully translocating through a NPC. The nuclear envelope bisects the field of view, running from top to bottom. The QD is observed arriving from the cytoplasm (left), residing at the NPC, and then leaving into the nucleus (right). (AVI 691 kb)
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Lowe, A., Siegel, J., Kalab, P. et al. Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking.Nature 467, 600–603 (2010). https://doi.org/10.1038/nature09285
- Received: 15 November 2009
- Accepted: 10 June 2010
- Published: 01 September 2010
- Issue Date: 30 September 2010
- DOI: https://doi.org/10.1038/nature09285
Editorial Summary
Nuclear pore complex selectivity
Nuclear pore complexes selectively transport cargoes across the nuclear envelope. Now, two studies published in this issue follow the movement of single cargo proteins (Lowe et al.) and mRNA transcripts (David Grünwald and Robert Singer) as they translocate across the nuclear envelope, with nanometre precision and high temporal resolution. They discover previously unidentified transport sub-steps and provide fundamental insight into the mechanism of selective transport through the nuclear pore complexes.