Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy

Abstract

The nuclear pore complex (NPC) perforates the nuclear envelope to facilitate selective transport between nucleus and cytoplasm. The NPC is composed of multiple copies of 30 different proteins, termed nucleoporins, whose arrangement within the NPC is an important unsolved puzzle in structural biology. Various alternative models for NPC architecture have been proposed but not tested experimentally in intact NPCs. We present a method using polarized fluorescence microscopy to investigate nucleoporin orientation in live yeast and mammalian cells. Our results support an arrangement of both yeast Nic96 and human Nup133–Nup107 in which their long axes are approximately parallel to the nuclear envelope plane. The method we developed can complement X-ray crystallography and electron microscopy to generate a high-resolution map of the entire NPC, and may be able to monitor nucleoporin rearrangements during nucleocytoplasmic transport and NPC assembly. This strategy can also be adapted for other macromolecular machines.

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Figure 1: Fluorescence anisotropy patterns reflect the orientation of fluorophores within the NPC.
Figure 2: Nic96-GFP constructs with a continuous linker α-helix.
Figure 3: Polarized fluorescence microscopy reveals anisotropy patterns for yeast strains expressing Nic96-GFP constructs.
Figure 4: Analysis of anisotropy patterns reveals approximate orientation of Nic96 within the NPC.
Figure 5: Anisotropy patterns in mammalian cells expressing Nup133-GFP.
Figure 6: Nup133-GFP anisotropy patterns are consistent with the published 'head-to-tail ring' arrangement of the Y-shaped subcomplex.

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Acknowledgements

We thank G. Blobel (Rockefeller University, Howard Hughes Medical Institute) for his generous support of this work, partially carried out in his laboratory. We thank D. Johnson and D. Muzzey for comments on the manuscript and C. Lue for technical assistance. M.K. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship. A.L.M. was supported by a Rockefeller University Women & Science Postdoctoral Fellowship. C.E.A., A.L.M. and S.M.S. were supported by US National Science Foundation grant BES-0620813 and National Institutes of Health grant R01 GM087977 to S.M.S.

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M.K., C.E.A. and A.L.M. did all experimentation, and M.K., C.E.A., A.L.M. and S.M.S. shared the design, analysis and writing.

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Correspondence to Martin Kampmann or Sanford M Simon.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 5939 kb)

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Kampmann, M., Atkinson, C., Mattheyses, A. et al. Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy. Nat Struct Mol Biol 18, 643–649 (2011). https://doi.org/10.1038/nsmb.2056

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