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In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport

Nature volume 467, pages 604–607 (2010)Cite this article

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Abstract

Export of messenger RNA occurs via nuclear pores, which are large nanomachines with diameters of roughly 120 nm that are the only link between the nucleus and cytoplasm1. Hence, mRNA export occurs over distances smaller than the optical resolution of conventional light microscopes. There is extensive knowledge on the physical structure and composition of the nuclear pore complex2,3,4,5,6,7, but transport selectivity and the dynamics of mRNA export at nuclear pores remain unknown8. Here we developed a super-registration approach using fluorescence microscopy that can overcome the current limitations of co-localization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nanometre precision. With this method we achieve 20-ms time-precision and at least 26-nm spatial precision, enabling the capture of highly transient interactions in living cells. Using this approach we were able to spatially resolve the kinetics of mRNA transport in mammalian cells and present a three-step model consisting of docking (80 ms), transport (5–20 ms) and release (80 ms), totalling 180 ± 10 ms. Notably, the translocation through the channel was not the rate-limiting step, mRNAs can move bi-directionally in the pore complex and not all pores are equally active.

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Figure 1: Super-registration precision and detection of nuclear mRNA.
Figure 2: Dwell times of β-actin mRNA at the NPC.
Figure 3: Binding sites of mRNAs at nuclear pores.
Figure 4: NPC topography of mRNA export.

References

  1. 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)

    Article  CAS  PubMed  Google Scholar 

  2. Kiseleva, E., Goldberg, M. W., Allen, T. D. & Akey, C. W. Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export. J. Cell Sci. 111, 223–236 (1998)

    CAS  PubMed  Google Scholar 

  3. Rout, M. P. et al. The yeast nuclear pore complex: Composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–652 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Beck, M. et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306, 1387–1390 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  5. 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  PubMed  Google Scholar 

  6. Alber, F. et al. The molecular architecture of the nuclear pore complex. Nature 450, 695–701 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Lim, R. Y. et al. Nanomechanical basis of selective gating by the nuclear pore complex. Science 318, 640–643 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  8. Mor, A. et al. Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells. Nature Cell Biol. 12, 543–552 (2010)

    Article  CAS  PubMed  Google Scholar 

  9. Stockley, P. G. et al. Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein. Nucleic Acids Res. 23, 2512–2518 (1995)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fusco, D. et al. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13, 161–167 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hallberg, E., Wozniak, R. W. & Blobel, G. An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin-like region. J. Cell Biol. 122, 513–521 (1993)

    Article  CAS  PubMed  Google Scholar 

  12. Cronshaw, J. A., Krutchinsky, A. N., Zhang, W. Z., Chait, B. T. & Matunis, M. J. Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158, 915–927 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  14. Nachury, M. V. & Weis, K. The direction of transport through the nuclear pore can be inverted. Proc. Natl Acad. Sci. USA 96, 9622–9627 (1999)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kopito, R. B. & Elbaum, M. Reversibility in nucleocytoplasmic transport. Proc. Natl Acad. Sci. USA 104, 12743–12748 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bachi, A. et al. The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. RNA 6, 136–158 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chakraborty, P., Satterly, N. & Fontoura, B. M. Nuclear export assays for poly(A) RNAs. Methods 39, 363–369 (2006)

    Article  CAS  PubMed  Google Scholar 

  18. Carmody, S. R. & Wente, S. R. mRNA nuclear export at a glance. J. Cell Sci. 122, 1933–1937 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Franke, W. W. & Scheer, U. The ultrastructure of the nuclear envelope of amphibian oocytes: a reinvestigation. II. The immature oocyte and dynamic aspects. J. Ultrastruct. Res. 30, 317–327 (1970)

    Article  CAS  PubMed  Google Scholar 

  20. Franke, W. W. & Scheer, U. The ultrastructure of the nuclear envelope of amphibian oocytes: a reinvestigation. I. The mature oocyte. J. Ultrastruct. Res. 30, 288–316 (1970)

    Article  CAS  PubMed  Google Scholar 

  21. Bastos, R., Pante, N. & Burke, B. Nuclear pore complex proteins. Int. Rev. Cytol. 162B, 257–302 (1995)

    CAS  PubMed  Google Scholar 

  22. Cordes, V. C., Reidenbach, S., Rackwitz, H. R. & Franke, W. W. Identification of protein p270/Tpr as a constitutive component of the nuclear pore complex-attached intranuclear filaments. J. Cell Biol. 136, 515–529 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ribbeck, K. & Gorlich, D. Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 20, 1320–1330 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Macara, I. G. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570–594 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  26. Lamond, A. I. & Spector, D. L. Nuclear speckles: a model for nuclear organelles. Nature Rev. Mol. Cell Biol. 4, 605–612 (2003)

    Article  CAS  Google Scholar 

  27. Kubitscheck, U. et al. Nuclear transport of single molecules: dwell times at the nuclear pore complex. J. Cell Biol. 168, 233–243 (2005)

    Article  PubMed  PubMed Central  Google Scholar 

  28. Clemen, A. E. et al. Force-dependent stepping kinetics of myosin-V. Biophys. J. 88, 4402–4410 (2005)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. 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  PubMed  PubMed Central  Google Scholar 

  30. Sun, C., Yang, W., Tu, L. C. & Musser, S. M. Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore. Proc. Natl Acad. Sci. USA 105, 8613–8618 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kubitscheck, U. et al. Nuclear transport of single molecules: dwell times at the nuclear pore complex. J. Cell Biol. 168, 233–243 (2005)

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank T. Dange, K. Czaplinski, T. Lionnet and X. Meng for help with cell lines and cloning; A. Gennerich, H. Y. Park and D. Entenberg for help on data analysis and programming; S. M. Shenoy for development of alignment software for detector pre-registration and his additional support; D. Larson, I. Lepper and U. Kubitscheck for providing fitting routines; and A. Wells for FISH and TIRF data, proof reading and discussion. Lentiviral vector was a gift from G. Mostoslavsky and R. C. Mulligan. Supported by DFG 3388/1 to D.G. Supported by NIH EB2060 and GM86217 to R.H.S. The authors express their appreciation to E. Gruss Lipper for her gift founding the Gruss Lipper Biophotonics Center that provided the equipment used in this research.

Author information

Authors and Affiliations

  1. Department of BioNanoScience, Kavli Institute of NanoScience, TU Delft, Lorentzweg 1, 2628 CJ Delft, The Netherlands,

    David Grünwald

  2. Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center 1300 Morris Park Avenue, Albert Einstein College of Medicine, Bronx, 10461, New York, USA

    David Grünwald & Robert H. Singer

Authors
  1. David Grünwald
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Contributions

D.G. designed and performed experiments, established cell lines, performed data analysis and built microscopy equipment in consultation with R.H.S. D.G. and R.H.S. discussed data and wrote the manuscript.

Corresponding author

Correspondence to Robert H. Singer.

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Supplementary information

Supplementary Information

This file contains Supplementary Results and Discussion, Supplementary References, Supplementary Tables 1-3 and Supplementary Figures 1-8 with legends. (PDF 746 kb)

Supplementary Movie 1

This movie shows β-actin mRNA scanning multiple pores - see Supplementary Information file for full legend. (MOV 66 kb)

Supplementary Movie 2

This movie shows the full length data set for a slow export event displayed in Fig. 1. panel K - see Supplementary Information file for full legend. (MOV 208 kb)

Supplementary Movie 3

This movie shows the fast export event from Fig. 1. panel M-P - see Supplementary Information file for full legend. (MOV 12 kb)

Supplementary Movie 4a

This movie shows mRNAs moving freely in the nucleoplasm and finally approaching nuclear pores at a low frequency. Several 'scanning' events can be visually identified - see Supplementary Information file for full legend. (MOV 2093 kb)

Supplementary Movie 4b

This movie shows mRNAs moving freely in the nucleoplasm and finally approaching nuclear pores at a low frequency. Several 'scanning' events can be visually identified displayed in real time at 50Hz - see Supplementary Information file for full legend. (MOV 2093 kb)

Supplementary Movie 5

This movie shows Animation of a 3D stack of POM121-tdTomato in the nuclear envelope - see Supplementary Information file for full legend. (MOV 3179 kb)

Supplementary Movie 6

This movie shows detection of mRNA without an NLS Signal on the MCP Label - see Supplementary Information file for full legend. (MOV 1149 kb)

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Grünwald, D., Singer, R. In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport. Nature 467, 604–607 (2010). https://doi.org/10.1038/nature09438

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