Formation of the postmitotic nuclear envelope from extended ER cisternae precedes nuclear pore assembly

Abstract

During mitosis, the nuclear envelope merges with the endoplasmic reticulum (ER), and nuclear pore complexes are disassembled. In a current model for reassembly after mitosis, the nuclear envelope forms by a reshaping of ER tubules. For the assembly of pores, two major models have been proposed. In the insertion model, nuclear pore complexes are embedded in the nuclear envelope after their formation. In the prepore model, nucleoporins assemble on the chromatin as an intermediate nuclear pore complex before nuclear envelope formation. Using live-cell imaging and electron microscope tomography, we find that the mitotic assembly of the nuclear envelope primarily originates from ER cisternae. Moreover, the nuclear pore complexes assemble only on the already formed nuclear envelope. Indeed, all the chromatin-associated Nup107-160 complexes are in single units instead of assembled prepores. We therefore propose that the postmitotic nuclear envelope assembles directly from ER cisternae followed by membrane-dependent insertion of nuclear pore complexes.

Figure 1.

Mitotic assembly of the nuclear envelope on the chromosome mass. Live-cell images were acquired from mitotic HeLa cells expressing GFP-Sec61β and H2B-mCherry. (A) 2D time-lapse series showing formation of the nuclear envelope on the chromosome mass (also see Video 1). Arrows indicate the rim of the chromosome mass, at which ER cisternae make initial contacts (0 s). Arrowheads follow the migrating edge of the nascent nuclear envelope from the rim to the center of the chromosome mass (120–420 s). The monochrome images highlight the nuclear envelope. (B) Assembly of the nuclear envelope followed in 3D. Deconvolved images from a mitotic cell acquired at various time points in 3D. The top row shows 2D images from the middle section. The dotted line labeled spindle axis connects the two spindle poles. The columns below show 3D renderings of the right chromosome mass together with the associated nuclear envelope. For clarity, only the bottom half of the chromosome mass is shown. The three time points (−240 s, 0 s, and 120 s) are from the same anaphase cell. The 0-s time point corresponds approximately to the onset of nuclear envelope assembly. The image set labeled completion is from another cell that has just completed nuclear envelope assembly. Views 1–3 show three regions on the chromosome mass surface. View 1 shows spindle pole proximal side; View 2 shows the rim; View 3 shows the spindle pole distal side. Bars, 10 µm.

Figure 2.

Transition during mitosis from ER cisternae to the nuclear envelope. (A and B) 2D time-lapse series from a mitotic HeLa cell expressing mCherry-Sec61β and LBR-GFP (A) or GFP-Rtn4HD (B) were acquired during mitotic nuclear envelope assembly. The tracings correspond to the normalized mean fluorescence intensity determined for Sec61β, LBR, and Rtn4HD in the masked regions representing peripheral ER and nascent nuclear envelope (NE). All data from A and B are representative results of three cells. norm., normalized. Bars, 10 µm.

Figure 3.

Effect during mitosis of spindle microtubules on nuclear envelope formation. 2D time-lapse images from mitotic HeLa cells expressing GFP-Sec61β and mCherry-tubulin were acquired during nuclear envelope assembly. (A) Control (also see Video 3) in the absence of nocodazole. (B) Nocodazole treated (also see Video 4). 33 µM nocodazole was added shortly after the onset of anaphase. Arrowheads indicate nuclear envelope initiation at the pole distal region of the chromosome mass. (C) Taxol treated (also see Video 5). 2 µM taxol was added shortly after the onset of anaphase. Arrowheads indicate a gap on the nuclear envelope obstructed by stabilized spindle microtubules. 0 s corresponds to the onset of nuclear envelope formation. Nuclear envelope (NE; green lines) corresponds to regions containing Sec61β on the contour of the chromosome mass, visible as a dark region because of its exclusion of soluble mCherry-tubulin. Bars, 10 µm.

Figure 4.

Direct contribution of ER cisternae to assembly of the nuclear envelope. (A and B) 3D renderings and serial optical sections show the relationship between the ER cisternae and nascent nuclear envelope in HeLa (A) and BSC1 (B) cells. 3D image stacks were acquired from mitotic cells expressing GFP-Sec61β and H2B-RFP. (a) Fluorescent image from the middle section. The boxed region contains an example of ER cisternae in continuity with the nascent nuclear envelope and is further shown in b together with evenly spaced serial images along the z axis. The section at 0.00 µm corresponds to the optical section in a. b′ displays rendered 3D views of region b. The example highlights a direct contact between ER cisternae (green lines) and the nascent nuclear envelope (NE; white lines). (C) EM tomograms of the nascent nuclear envelope from an anaphase BSC1 cell. (a) Low magnification image of the whole cell. (b and c) A representative EM tomographic slice from the boxed region is shown without (b) and with (c) superimposition of a model for ER cisterna (green) and ribosomes (magenta). Chrom., chromosome mass. (d–f) Three views of the rendered 3D model. ER membrane, ribosome, and the surface of the chromosome mass are colored in green, magenta, and blue, respectively. Bars: (A–C, a) 10 µm; (A and B, b) 2 µm; (C, b and f) 200 nm.

Figure 5.

Assembly of higher order Nup133 structures is restricted to the nascent nuclear envelope membrane. (A) Association of Nup133, a component of the Nup107–160 complex, and Nup62 with the chromosome mass during early anaphase. The image is the middle section of a HeLa cell coexpressing GFP×3-Nup133 and H2B-mCherry (left) or GFP-Nup62 and H2B-mCherry (right) 30 s before its recruitment onto the nuclear envelope. The signal of H2B-mCherry (not depicted) was used to delineate the outer contours of the chromosome masses (dotted red lines). (B) Mean fluorescence intensity ratios ± SD of chromosome mass to cytosol for Nup133 and Nup62. The difference in ratios is statistically significant (P = 0.008). Chrom., chromosome. (C) A 2D time lapse from a mitotic HeLa cell expressing GFP×3-Nup133 and mCherry-Sec61β showing the recruitment of Nup133 onto the nuclear envelope (also see Video 6). The boxed regions are enlarged in D, and further enlarged boxed regions from D are shown in E. The onset of nuclear envelope formation is at 0 s. Open arrowheads show Nup133 associated with kinetochores. Closed arrowheads show Nup133 recruited to the nascent nuclear envelope. Note that Nup133 is absent on the surface of the chromosome mass at sites devoid of the nuclear envelope. Bars: (A and C) 10 µm; (D and E) 5 µm.

Figure 6.

Assembly of higher order Nup107–160 structures, visualized by imaging Seh1 or Nup37, is restricted to the nascent nuclear envelope membrane. (A) 2D time lapse from a mitotic HeLa cell coexpressing GFP-Seh1 and mCherry-Sec61β showing the recruitment of Seh1 (closed arrowheads) onto the nuclear envelope. (B) 2D time lapse from a mitotic HeLa cell coexpressing GFP-Nup37 and mCherry-Sec61β showing the recruitment of Nup37 (closed arrowheads) onto the nuclear envelope. Open arrowheads indicate kinetochores. See legend of Fig 5 (C and D) for a detailed description. Boxes in A and B are enlarged at the bottom. Bars, 10 µm.

Figure 7.

Quantification of GFP×3-Nup133 during the postmitotic nuclear pore assembly. HeLa cells coexpressing GFP×3-Nup133 and H2B-mCherry (not depicted) were chemically fixed and then imaged in 3D. Contours around the chromosome masses and nuclei were established by following the location of the H2B-mCherry fluorescence signal (dotted red lines). (A) Interphase nuclear pores. The image is from a nuclear envelope located at the bottom side of nucleus close to the glass coverslip. Fluorescence intensity distribution of 1,690 GFP×3-Nup133 spots imaged in 16 cells. The data represent the results from 16 cells. (B) Image from the middle section of a cell in anaphase acquired during the initial stages of Nup133 recruitment. The yellow boxes contain diffraction-limited fluorescent spots acquired from (a) rim region, (b) kinetochore region, (c) chromosome mass, (d) cytosol, and (e) control region without objects (background). Nondiffraction-limited objects (arrowheads) were excluded from the analysis. (C) Schematic representation of the regions used for analysis. (D) Fluorescence intensity distribution of diffraction-limited spots from each of the four regions color coded as indicated in C. (E) Image from the middle section of a cell in anaphase before the onset of Nup133 recruitment to the nuclear envelope and fluorescence intensity distribution of diffraction-limited Nup133 spots at the rim of the chromosome mass. (F) Image from the middle section of a cell in telophase and fluorescence intensity distribution of diffraction-limited Nup133 spots at the rim of the chromosome mass. Note the appearance of a population of spots peaking at approximately four GFP×3-Nup133. In B–D, the data represent the results from three cells. Chrom., chromosome. Bars, 10 µm.

Figure 8.

Postmitotic import of IBB, summary of kinetic results obtained in this study, and working model for the postmitotic assembly of the nuclear envelope and nuclear pores. (A) 2D time lapse of a mitotic HeLa cell expressing GFP-Sec61β and IBB-tomato acquired during nuclear envelope assembly (also see Video 7). Anaphase onset is at 0 s. At 660 s, imported IBB was clearly visualized next to the rim of the chromosome mass (arrows), at a time when assembly of the nuclear envelope is still incomplete as highlighted by the gap between the arrowheads. Key events were obtained by quantification of the Sec61β and IBB fluorescence signals. Bar, 10 µm. (B) Schematic summary of the relative kinetics of nuclear envelope formation and nucleoporin recruitment. The plots represent the fluorescence intensity associated with formation of the nuclear envelope (Sec61β, green; Fig. S1 B), recruitment of Nup133 (yellow; Fig. S1 C), recruitment of Nup62 (blue; Fig. S1 D), and import of IBB (red; Fig. S1 E). (C) Working model for the postmitotic assembly of the nuclear envelope and nuclear pores. ER is shown in light green; nuclear envelope (NE) is shown in dark green; yellow dot shows Nup107–160 complex; magenta dot shows remaining component of the nuclear pore; red square shows IBB; Chrom., chromosome mass.