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

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.

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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

Authors

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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The authors declare no competing financial interests.

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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