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Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings

Nature Structural & Molecular Biology volume 25, pages 21–28 (2018)Cite this article

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Abstract

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.

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Fig. 1: The NE forms from highly fenestrated ER sheets.
Fig. 2: NPC assembly proceeds by dilation of small membrane holes.
Fig. 3: Small ER and NE holes with similar diameters to those of prepores in cells at early time points.
Fig. 4: Intensity distribution inside the prepore membranes and unbiased classification of prepores from spectral ordering.
Fig. 5: Structural maturation of prepores revealed by subtomogram averaging and postmitotic NE and NPC assembly model.
Fig. 6: Live imaging of genome-edited cells.

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References

  1. Beck, M. & Hurt, E. The nuclear pore complex: understanding its function through structural insight. Nat. Rev. Mol. Cell Biol. 18, 73–89 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Ungricht, R. & Kutay, U. Mechanisms and functions of nuclear envelope remodelling. Nat. Rev. Mol. Cell Biol. 18, 229–245 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Weberruss, M. & Antonin, W. Perforating the nuclear boundary - how nuclear pore complexes assemble. J. Cell Sci. 129, 4439–4447 (2016).

    CAS  PubMed  Google Scholar 

  4. LaJoie, D. & Ullman, K. S. Coordinated events of nuclear assembly. Curr. Opin. Cell Biol. 46, 39–45 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Dultz, E. et al. Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J. Cell Biol. 180, 857–865 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Otsuka, S., Szymborska, A. & Ellenberg, J. Imaging the assembly, structure, and function of the nuclear pore inside cells. Methods Cell Biol. 122, 219–238 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Rabut, G., Lénárt, P. & Ellenberg, J. Dynamics of nuclear pore complex organization through the cell cycle. Curr. Opin. Cell Biol. 16, 314–321 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Antonin, W., Ellenberg, J. & Dultz, E. Nuclear pore complex assembly through the cell cycle: regulation and membrane organization. FEBS Lett. 582, 2004–2016 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Wandke, C. & Kutay, U. Enclosing chromatin: reassembly of the nucleus after open mitosis. Cell 152, 1222–1225 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Schellhaus, A. K., De Magistris, P. & Antonin, W. Nuclear reformation at the end of mitosis. J. Mol. Biol. 428 10 Pt A, 1962–1985 (2016).

    Article  Google Scholar 

  11. Fichtman, B., Ramos, C., Rasala, B., Harel, A. & Forbes, D. J. Inner/outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis. Mol. Biol. Cell 21, 4197–4211 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lu, L., Ladinsky, M. S. & Kirchhausen, T. Formation of the postmitotic nuclear envelope from extended ER cisternae precedes nuclear pore assembly. J. Cell Biol. 194, 425–440 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Otsuka, S. et al. Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope. eLife 5, e19071 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Walther, T. C. et al. The conserved Nup107-160 complex is critical for nuclear pore complex assembly. Cell 113, 195–206 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Rotem, A. et al. Importin beta regulates the seeding of chromatin with initiation sites for nuclear pore assembly. Mol. Biol. Cell 20, 4031–4042 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Puhka, M., Joensuu, M., Vihinen, H., Belevich, I. & Jokitalo, E. Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells. Mol. Biol. Cell 23, 2424–2432 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Goldberg, M. W., Wiese, C., Allen, T. D. & Wilson, K. L. Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly. J. Cell Sci. 110, 409–420 (1997).

    CAS  PubMed  Google Scholar 

  18. Kiseleva, E., Rutherford, S., Cotter, L. M., Allen, T. D. & Goldberg, M. W. Steps of nuclear pore complex disassembly and reassembly during mitosis in early Drosophila embryos. J. Cell Sci. 114, 3607–3618 (2001).

    CAS  PubMed  Google Scholar 

  19. Kosinski, J. et al. Molecular architecture of the inner ring scaffold of the human nuclear pore complex. Science 352, 363–365 (2016).

    Article  CAS  PubMed  Google Scholar 

  20. Vollmer, B. et al. Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly. EMBO J. 31, 4072–4084 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Eisenhardt, N., Redolfi, J. & Antonin, W. Interaction of Nup53 with Ndc1 and Nup155 is required for nuclear pore complex assembly. J. Cell Sci. 127, 908–921 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Champion, L., Linder, M. I. & Kutay, U. Cellular reorganization during mitotic entry. Trends Cell Biol. 27, 26–41 (2017).

    Article  PubMed  Google Scholar 

  23. Lukinavičius, G. et al. SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat. Commun. 6, 8497 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Narayan, K. et al. Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology. J. Struct. Biol. 185, 278–284 (2014).

    Article  CAS  PubMed  Google Scholar 

  25. Cardona, A. et al. TrakEM2 software for neural circuit reconstruction. PLoS One 7, e38011 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Fehrenbach, J., Weiss, P. & Lorenzo, C. Variational algorithms to remove stationary noise: applications to microscopy imaging. IEEE Trans. Image Process. 21, 4420–4430 (2012).

    Article  PubMed  Google Scholar 

  27. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  28. Sommer, C., Straehle, C., Koethe, U. & Hamprecht, F. A. ilastik: Interactive Learning and Segmentation Toolkit. in Proc. of the IEEE International Symposium on Biomedical Imaging 230‒233 (IEEE, Piscataway, New Jersey, 2011).

  29. Belevich, I., Joensuu, M., Kumar, D., Vihinen, H. & Jokitalo, E. Microscopy image browser: a platform for segmentation and analysis of multidimensional datasets. PLoS Biol. 14, e1002340 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  31. De Lathauwer, L., De Moor, B. & Vandewalle, J. A multilinear singular value decomposition. SIAM J. Matrix Anal. Appl. 21, 1253–1278 (2000).

    Article  Google Scholar 

  32. Kolda, T. G. & Bader, B. W. Tensor decompositions and applications. SIAM Rev. 51, 455–500 (2009).

    Article  Google Scholar 

  33. Atkins, J. E., Boman, E. G. & Hendrickson, B. A spectral algorithm for seriation and the consecutive ones problem. SIAM J. Comput. 28, 297–310 (1998).

    Article  Google Scholar 

  34. Ding, C. & He, X. Linearized cluster assignment via spectral ordering. in Proceedings of the 21st International Conference on Machine Learning 30 (2004).

  35. Mahen, R. et al. Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells. Mol. Biol. Cell 25, 3610–3618 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Koch, B. et al. Generation and validation of homozygous fluorescent knock-in cells using genome editing. Preprint at https://doi.org/10.1101/188847 (2017).

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Acknowledgements

We thank the European Molecular Biology Laboratory Electron Microscopy Core Facility, the members of the Ellenberg group and the Beck group for advice and discussion, and A. Kreshuk and I. Belevich for help with using Ilastik and MIB, respectively. This work was supported by grants from the German Research Council to J.E. (DFG EL 246/3-2 within the priority program SPP1175), the Baden-Württemberg Stiftung to J.E., and the European Research Council to M.B. (309271-NPCAtlas), as well as by the European Molecular Biology Laboratory (S.O., A.M.S., M.S., J.K.H., M.J.H., S.S., Y.S., M.B., and J.E.). S.O. was additionally supported by the EMBL Interdisciplinary Postdoc Programme (EIPOD) under Marie Curie Actions COFUND and a JSPS fellowship (The Japan Society for the Promotion of Science, postdoctoral fellowship for research abroad).

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Authors and Affiliations

  1. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Shotaro Otsuka, Anna M. Steyer, Jean-Karim Hériché, M. Julius Hossain, Suruchi Sethi, Moritz Kueblbeck, Yannick Schwab, Martin Beck & Jan Ellenberg

  2. Electron Microscopy Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany

    Martin Schorb & Yannick Schwab

  3. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Martin Beck

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  1. Shotaro Otsuka
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Contributions

S.O., A.M.S., Y.S., M.B., and J.E. designed the project. S.O. performed quantitative analysis of FIB-SEM data and all of the experiments and analyses of EM tomography. A.M.S. acquired all of the FIB-SEM data and carried out segmentation of FIB-SEM images. M.S. contributed to the computational quantitative analysis of EM images. J.-K.H. performed spectral ordering. M.J.H. carried out the segmentation of fluorescence images and assisted with computational analysis of EM images. S.S. helped with the segmentation of FIB-SEM images. M.K. generated genome-edited cell lines. Y.S., M.B., and J.E. supervised the work. S.O. and J.E. wrote the paper. All authors contributed to the analysis and interpretation of data and provided input on the manuscript.

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Correspondence to Jan Ellenberg.

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Integrated Supplementary Information

Supplementary Figure 1 Live-cell and EM images of cells analyzed by FIB-SEM (a) and EM tomography (b).

Cells cultured on sapphire disks were imaged by widefield microscopy (upper panels). After high-pressure freezing and plastic-embedding, the same cells were observed by SEM or TEM. The contrast of SEM images shown in (a) are enhanced by projection of a series of 20 images for presentation purposes. TEM images shown in (b) are from one of the serial sections of 300 nm thickness. Time after anaphase onset (AO) is indicated. Scale bars, 10 μm.

Supplementary Figure 2 Galleries of postmitotic NPC assembly intermediates and quantification of membrane distance and curvature.

(a) Other images of pre-pores at different times after AO (4.8, 6.1, 7.7, 10, and 15 min). ONM, outer nuclear membrane; INM, inner nuclear membrane. Scale bar, 100 nm. (b, c) Quantification of the distance between ONM and INM (b) and the radius of membrane tip curvature (c) as indicated by a blue bidirectional arrow and a red arrow in the left panels. The plots of the ONM/INM distance and the tip curvature are from 71, 75, 88, 64, 58, and 52 pores at 4.8, 6.1, 7.7, 10, 15 min, and in interphase, respectively. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. A blue line shows a fitted sigmoid curve. For comparison, a sigmoid curve of pre-pore diameter in Fig. 2c is shown by a pink dashed line. Interestingly, the distance between ONM and INM was very highly variable during early stages of NPC assembly, and became constrained from 36 to 25 nm from 10 min onwards only after pore dilation was completed (b), suggesting that the linker of nucleoskeleton and cytoskeleton (LINC) complex, which connects ONM and INM (Shimi, T., Butin-Israeli, V. & Goldman, R. D. Curr. Opin. Cell Biol. 24, 71–78, 2012), is established after NPC assembly is completed. On the other hand, the tip curvature of the pre-pore membrane was constant at all time points (c), implying the continuous association of membrane-shaping protein(s) such as reticulons followed by Nups155 and 53 during NPC assembly3.

Supplementary Figure 3 Distribution of nuclear pore diameter measured in Fig. 2b.

The diameter of pre- and mature pores at different times after AO is shown in histograms. The diameter of total nuclear pores is also plotted in a histogram and fitted with two Gaussian functions. The intersection of two Gaussians was 51.3 nm, and this value was used to define smaller and larger pre-pores for Fig. 2d.

Supplementary Figure 4 Nuclear surface area measurement for normalizing nuclear pore density.

(a) Time-lapse three-dimensional imaging of dividing HeLa cells which express histone-H2b-mCherry. Images show single confocal sections (upper panels) and the segmented nuclei (lower panels) at indicated times after AO. Scale bar, 10 μm. (b) Quantification of nuclear surface area. Black dots and gray lines represent the average and s.d. of measurements from 19 nuclei. (c) The nuclear surface area measured in (a, b) and the density of small NE holes, pre-pores smaller and larger than 51 nm, and mature pores at different times measured by electron tomography (the data are summarized in Supplementary Table 2). The pores at later than 19 min are defined as mature pores. The pore density at 19–60 min is the average from 6 cells at 19, 24, 28, 36, 42, and 53 min after AO, which was measured previously 13. The surface area of the nucleus at each time point is plotted above the density histogram. At each time point the density was multiplied by the nuclear surface area to remove the effect of nuclear expansion on the pore density.

Supplementary Figure 5 Individual radial profiles of prepores shown in Fig. 4a.

Bold and fine lines represent the average and s.d. of measurements from 72, 78, 88, 64, 58, and 52 pores at 4.8, 6.1, 7.7, 10, 15 min, and in interphase, respectively. The position of the membrane wall is indicated by a short line on the X-axis for each time point.

Supplementary Figure 6 Seriation contingency table.

Fraction of nuclear pores at different times after AO is color-coded in each cluster. Cluster 1, 2, 3, 4, and 5 contains the pores at the rank of 1–61, 62–146, 147–215, 216–323, 324–399, respectively.

Supplementary Figure 7 3D structural comparison of chronologically ordered prepores and mature pores.

Electron tomographic slices of single (a) and averaged (b) pre- and mature pores at indicated time points are shown. No symmetry was imposed in averaging. The averaged images are from 72, 78, 86, 64, 58, 52 pores for 3.1, 3.9, 4.3, 5.3, 5.7, and 6.3 min, respectively. Red arrowheads i, ii, and iii on side-view images indicate the locations of the planes which are inclined at 90° in top views i, ii, and iii. Scale bars, 100 nm. (c) Quantification of the inner ring intensity. As in Fig. 4a, the region indicated by red dashed box in the left panel was Z-projected and the radial intensity from the center of the pore was measured. Intensity was normalized to that of NE lumen. The intensity in the region adjacent to the membrane as indicated by a green line in the left panels was quantified and plotted. The plot shows the mean from 71, 75, 88, 64, 58, and 52 pores at 4.8, 6.1, 7.7, 10, 15 min, and in interphase, respectively. A green line shows a sigmoid curve. For comparison, a sigmoid curve of pre-pore diameter in Fig. 2c is shown by a pink dashed line.

Supplementary Figure 8 Southern blotting of genome-edited mEGFP-Nup205 cell clones.

Genomic DNA of each cell clone was digested with restriction enzymes and the fragments were detected by probes for Nup205 (a) and GFP (b). The size of the fragments, restriction enzyme (RE) digestion sites, and the probe-binding regions are illustrated in the bottom panels. The clone #81 showed only a band at 4.0 kb whereas the other clones exhibited additional bands at 3.2 kb (a), indicating a homozygous knock-in for the clone #81 and a heterozygous knock-in for the others. The GFP probe did not recognize any extra bands (b), suggesting no extra GFP insertion into the other region of the genome.

Supplementary information

Videos

Supplementary Video 1

Focused ion beam scanning electron microscopy (FIB-SEM) of a whole cell at 6.3 min after anaphase onset. Only 20% of FIB-SEM slices were shown in order to reduce the size of this movie. For full segmentation, the chromosome (light blue), the ER membranes in proximity to the chromosome (dark blue), and prepores (yellow) were segmented every 10–15th slice, and the slices in between were interpolated. Scale bar, 2 μm

Supplementary Video 2

Fine segmentation of the nuclear envelope (dark blue) and prepores of a part of a cell at 6.3 min after anaphase onset. The prepore is indicated by a yellow sphere with the average prepore diameter, and the center of the sphere is depicted by a yellow dot. The segmentation was done in every single slice. The upper part shows FIB-SEM images and the bottom part shows the ones with the segmentation. C, cytoplasm; N, nucleoplasm. Scale bar, 1 μm

Supplementary Video 3

Electron tomographic slices of the nuclear envelope of a cell at 7.7 min after anaphase onset. Prepores are indicated by pink arrows. C, cytoplasm; N, nucleoplasm. Scale bar, 100 nm

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Otsuka, S., Steyer, A.M., Schorb, M. et al. Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings. Nat Struct Mol Biol 25, 21–28 (2018). https://doi.org/10.1038/s41594-017-0001-9

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