Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope
- European Molecular Biology Laboratory, Germany
Figures
Figure 1 with 3 supplements
Interphase assembly intermediates of nuclear pore complexes (NPCs).
(A) Correlative live-cell imaging with electron microscopy (EM). Cell-cycle progression of HeLa cells was monitored by confocal microscopy and the same cell was subjected to electron tomography. …
Figure 1—figure supplement 1
Estimation of core regions.
(A,B) Time-lapse three-dimensional (3D) imaging of dividing HeLa cells which express Lap-2α-YFP and histone-H2b-mCherry by confocal microscopy. Scale bars, 10 µm. (A) Maximum intensity projection …
Figure 1—figure supplement 2
Live-cell and EM images of cells analyzed by EM tomography.
Cells cultured on carbon-patterned sapphire disks were imaged by confocal microscopy (upper panels). After high-pressure freezing, plastic-embedding, and serial sectioning, the same cells were …
Figure 1—figure supplement 3
Galleries of interphase NPC assembly intermediates.
(A) Other images of immuno-EM in Figure 1C. Assembly intermediates are indicated by white arrows. The profile of the nuclear envelope (NE) and the positions of gold particles in one EM image are …
Figure 2
Quantitative structural comparison of assembly intermediates.
(A) Electron tomographic slices of assembly intermediates in cells captured at 53, 28, and 19 min after anaphase onset (AO) and ONM/INM fusion events. Profiles of ONM (gray) and INM (blue) in black …
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Figure 2—source data 1
Depth and diameter values used for Figure 2C–F.
- https://doi.org/10.7554/eLife.19071.010
Figure 3 with 2 supplements
Abundance of mature pores and assembly intermediates at different cell-cycle stages.
(A) Measurement of nuclear pore density. Gray sheets are the NEs segmented from EM tomograms. Blue and red dots indicate the positions of mature pores and intermediates, respectively. Inner-core …
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Figure 3—source data 1
Density values used for Figure 3B–D.
- https://doi.org/10.7554/eLife.19071.012
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Figure 3—figure supplement 2—source data 1
Surface area values used for Figure 3— figure supplement 2C.
- https://doi.org/10.7554/eLife.19071.013
Figure 3—figure supplement 1
Modeling the density of nuclear pores.
(A) Reaction scheme of the delay equation model (Equations 1‒3). V, production rate; , maturation initiation rate constant; , maturation delay; , degradation rate constant. The details are …
Figure 3—figure supplement 2
Nuclear surface area measurement for the modeling.
(A) Time-lapse 3D imaging of dividing HeLa cells which express histone-H2b-mCherry (indicated in red). Images show single confocal sections (upper) and the segmented nuclei (lower) at indicated time …
Figure 4 with 1 supplement
Live imaging of nuclear pore assembly in core regions.
(A) Time-lapse three-dimensional (3D) imaging of GFP-Nup107 and GFP-Nup358 genome-edited cells. DNA was stained with silicon–rhodamine (SiR) Hoechst (Lukinavicius et al., 2015). Single confocal …
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Figure 4—source data 1
Intensity values used for Figure 4B,C.
- https://doi.org/10.7554/eLife.19071.017
Figure 4—figure supplement 1
Characterization of genome-edited cell lines expressing GFP-Nup107 and GFP-Nup358.
(A) Junction PCR of GFP-Nup107 (left) and GFP-Nup358 (right) cells with forward primers annealing the upstream of Nups and reverse primers annealing inside of Nups as indicated by black arrows in …
Figure 5
Stimulated emission depletion (STED) imaging of assembly intermediates.
GFP-Nup107 genome-edited cells were stained with anti-GFP and anti-Nup358 antibodies. (A) STED images of cells at 24 and 108 min after anaphase onset. Scale bar, 10 µm. (B) Flattened and enlarged …
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Figure 5—source data 1
Frequency values used for Figure 5C.
- https://doi.org/10.7554/eLife.19071.020
Figure 6 with 1 supplement
3D structural comparison of assembly intermediates.
(A,B) Electron tomographic slices of single (A) and averaged (B) mature pores and intermediates at selected time points (53, 28, and 19 min). The averaged images are from 36 mature pores and 14, 11, …
Figure 6—figure supplement 1
Stability of the subtomogram averaging.
(A) Illustration of the intensity profile analysis. Images of the averaged assembly intermediate at 53 min post anaphase are shown as an example. Red arrowheads and a dotted line in the side view …
Videos
Video 1
EM tomographic slices of the nuclear envelope of a cell at 53 min post anaphase.
One of the mature pores and an assembly intermediate are indicated by blue and red arrows, respectively. Scale bar, 100 nm.
Tables
Table 1
Summary of EM tomography. A data table shows the surface area of the NE analyzed by EM tomography and the number of mature pores, assembly intermediates, and the outer and inner nuclear membrane …
| Time after anaphase onset (min) | 19.2 | 24.4 | 28.4 | 36.3 | 42.0 | 53.2 | 61.0 | 65.6 | 73.6 | 82.9 | 100 | 116 | >180 | >180 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Non-core | Analyzed surface area (µm2) | 5.29 | 4.04 | 5.06 | 3.99 | 5.17 | 5.54 | 4.04 | 3.72 | 3.06 | 3.73 | 3.32 | 3.17 | 4.03 | 2.80 |
| Number of mature pores | 82 | 45 | 48 | 42 | 62 | 51 | 53 | 33 | 33 | 39 | 33 | 31 | 45 | 31 | |
| Number of intermediates | 2 | 4 | 6 | 3 | 5 | 8 | 3 | 3 | 2 | 1 | 5 | 5 | 4 | 1 | |
| Number of ONM/INM fusion | |||||||||||||||
| Inner-core | Analyzed surface area (µm2) | 5.77 | 5.06 | 4.16 | 4.74 | 6.75 | 4.33 | 3.61 | 4.57 | 4.07 | 3.53 | 4.61 | 3.46 | ||
| Number of mature pores | 24 | 16 | 3 | 18 | 30 | 16 | 34 | 27 | 27 | 39 | 53 | 32 | |||
| Number of intermediates | 21 | 13 | 12 | 8 | 35 | 12 | 1 | 3 | 4 | 3 | 2 | 3 | |||
| Number of ONM/INM fusion | 2 | 1 | |||||||||||||
| Outer-core | Analyzed surface area (µm2) | 4.22 | 4.15 | 3.83 | 2.55 | 4.78 | 4.41 | 3.59 | 3.68 | 2.71 | 2.67 | 2.77 | 2.68 | ||
| Number of mature pores | 29 | 40 | 27 | 17 | 44 | 35 | 38 | 28 | 30 | 34 | 25 | 28 | |||
| Number of intermediates | 23 | 10 | 19 | 9 | 13 | 11 | 1 | 4 | 1 | 3 | 5 | 1 | |||
| Number of ONM/INM fusion | 2 |
