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First published online January 23, 2008
doi: 10.1242/10.1242/jcs.022525

Journal of Cell Science 121, 317-328 (2008)
Published by The Company of Biologists 2008
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A role for gp210 in mitotic nuclear-envelope breakdown

Vincent Galy1,*,{ddagger},§, Wolfram Antonin1,{ddagger},, Andreas Jaedicke1, Martin Sachse2, Rachel Santarella1, Uta Haselmann1 and Iain Mattaj1,§

1 EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
2 Plateforme de microscopie électronique, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris CEDEX 15, France


Figure 1
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  		Fig. 1. gp210-dsRNA-mediated depletion or mutation leads to formation of twinned nuclei. (A) Two-cell-stage embryos from control(RNAi), gp210(RNAi) or gp210(tm2320) nematodes were observed by DIC microscopy. Bar, 10 µm. (B) Total protein extracts from N2 (lane 1) or gp210(tm2320) (lane 2) nematodes were separated by SDS PAGE, transferred and probed with antibodies against gp210 or NUP107. (C) gp210 (green in merge) and NPCs labeled with mAb414 (red in merge) in fixed embryos expressing GFP-LEM2 (top) were observed by epifluorescence (dsRNA-treated embryos) or confocal microscopy [GFP::lem-2 and gp210(tm2320),GFP::lem-2 embryos)]. Bars, 10 µm. (D) gp210(RNAi) embryos were fixed by high-pressure freezing in utero and observed by TEM. Overview picture (left) of twinned nuclei and a higher magnification (right) of a normal NE containing NPCs (arrows). Note the presence of a stacked NE (arrowhead). Bars, 1 µm (overview) and 100 nm (zoom).

 

Figure 2
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  		Fig. 2. gp210-dsRNA-mediated depletion or mutation affects lamin depolymerization and chromosome mixing. (A) Confocal still images from time-lapse recordings of the first zygotic division of control(RNAi) (top) and gp210(RNAi) (bottom) embryos expressing YFP-lamin and GFP–β-tubulin (not recorded). Recordings were synchronized relative to the onset of anaphase. Abnormal YFP-lamin remaining during mitosis (arrowheads) and the formation of twinned daughter nuclei (arrows) are indicated. Bar, 10 µm. (B) Confocal still images from time-lapse recordings of the first zygotic division of gp210(RNAi) embryos expressing GFP-histone H2B show that the maternal and paternal chromosomes remain separated during mitosis (arrowheads) and are packaged in two distinct nuclei after mitosis. Bar, 5 µm. (C) TEM image of a twinned nucleus (arrows) derived from a two-cell-stage gp210(tm2320) embryo entering metaphase. Condensed chromosomes (top panel) are partially aligned but physically separated by two NEs (arrowheads). Bar, 500 nm. At a higher magnification (bottom panel), spindle microtubules (short arrows) are visibly aligned towards the chromosomes, and the nucleoplasmic and cytoplasmic components are mixed, indicated by the ribosomes (black dots) seen in the nucleoplasm. Note the stacked membrane segments of the NE (long arrows). Bar, 100 nm. (D) gp210(RNAi) embryos expressing YFP-lamin (green in merge) were processed for the immunolocalization of NPCs with mAb414 (red in merge) and NUP153 (not in merge) together with chromatin (blue in merge) and observed by epifluorescence microscopy. The remaining lamins of the ongoing (arrowheads) or the previous cell division (arrows) are indicated. Bars, 2 µm.

 

Figure 3
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  		Fig. 3. gp210-dsRNA-mediated depletion or mutation affects lamin depolymerization and chromosome mixing. (A) Confocal still images from time-lapse recordings of the first zygotic division of control(RNAi) (top) and gp210(RNAi) (bottom) embryos expressing GFP-emerin. Recordings were synchronized relative to the onset of anaphase. Abnormal GFP-emerin remaining during mitosis (arrowheads) as well as close to the centrosomes (indicated by stars, see also supplementary material Movie 1). Bars, 10 µm. (B) Control(RNAi) and gp210(RNAi) embryos were fixed by high-pressure freezing in utero and observed by TEM. Overview pictures (top) and a detailed view (bottom) of the NE at prometaphase (left) and telophase (right) are shown. A stacked NE (arrowheads) was seen in both dsRNA-treated samples at prometaphase with aligned NPCs (arrow) but only in gp210(RNAi) embryos during telophase. These stacks are different from endoplasmic reticulum (ER) as they have ribosomes on one face only and possess a constant lumen size. N, nucleus; Ch, chromatin; *, centrosome. Bars, 1 µm (overviews) and 200 nm (zoom, middle and lowest prometaphase panels and the lowest telophase panels). (C) Confocal still images from time-lapse recordings of the first zygotic divisions showing the effect of gp210 and lamin co-depletion through dsRNA treatment at the two-cell-stage (top) or four-cell-stage (bottom) of embryos expressing GFP-LEM2. Young adults were injected with gp210 dsRNAs and fed with bacteria expressing either control (left) or lamin (right) dsRNA. The twinned nuclei (arrows) induced upon gp210-dsRNA-mediated depletion were absent when lamin was co-depleted. The single nuclei visible were sometimes misshapen (arrowheads), owing to depletion of the lamin. Bar, 10 µm.

 

Figure 4
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  		Fig. 4. gp210 is enriched in the NE and its remnants during NEBD. Wild-type embryos were fixed, processed for immunostaining for gp210 (green in merge) or NPCs (using mAb414, red in merge) together with Hoechst chromatin staining (blue in merge) and observed by confocal microscopy. gp210 remained associated with the mitotic NE (arrowheads in metaphase and anaphase), whereas NPCs were largely disassembled (mAb414). The intensity of the gp210 signal was even stronger during metaphase than during interphase or telophase. Bar, 10 µm.

 

Figure 5
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  		Fig. 5. NEBD is inhibited by Fab fragments against the C-terminal domain of gp210. (A) Nuclei were assembled in the presence of either control Fab fragments against the lumenal domain of gp210 or inhibitory Fab fragments against the C-terminal domain of gp210, respectively. After two hours, a twofold volume of mitotic extract was added and the samples incubated for another 2 hours. Membranes were stained by DiIC18, chromatin with DAPI and samples analyzed by confocal microscopy. (B) An experiment was performed as in A except that, instead of mitotic extracts, cyclin B {Delta}90 was added to drive the system into mitosis. (C) An experiment was performed as in A except that the Fab fragments against the indicated nucleoporins were added after 110 minutes and mitotic extract after 120 minutes. (D) An experiment was performed as in A except that samples were analyzed using antibody against NUP153 (green) and mAb414 (red). Chromatin was stained with DAPI (blue in overlays). (E) An experiment was performed as in A except that samples were analyzed using an antibody against phospho-histone H3 (green) and the monoclonal antibody mAb414 (red). Chromatin was stained with DAPI (blue in upper row). Bars, 10 µm.

 

Figure 6
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  		Fig. 6. The interphase function is not blocked by Fab fragments against the C-terminal domain of gp210. (A) Nuclei were assembled in the presence of either control Fab fragments against the lumenal domain of gp210 or inhibitory Fab fragments against the C-terminal domain of gp210. After 30 minutes, biotinylated-dUTP was added. After 90 minutes, replication foci were visualized with Alexa-488-labeled streptavidin, and chromatin visualized with DAPI. Control samples in the lower row were treated with aphidicolin. (B) Nuclei were assembled as in A. After 90 minutes, a fluorescently labeled import substrate (upper row) or a control substrate (lower row) were added. The reaction was stopped after 30 additional minutes by fixation and isolated. Chromatin is stained with DAPI (blue) and membranes with DiIC18 (red). (C) Nuclei were assembled as in A. After 90 minutes, a biotinylated nondegradable cyclin B mutant protein was added. The reaction was stopped after 30 additional minutes by fixation and isolated. Biotinylated cyclin B was visualized with fluorescently labeled streptavidin (green) and NPCs by means of mAb414 staining (red). Bars, 10 µm.

 

Figure 7
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  		Fig. 7. Fab fragments against the C-terminal domain of gp210 block mitotic phosphorylation. Membranes were pre-incubated with no, control or inhibitory Fab fragments. Interphase or mitotic extracts and radioactive [{gamma}-32P]ATP were added and incubated for 20 minutes. The reaction was stopped by solubilization of the membranes, gp210 was immunoprecipitated and the samples analyzed by autoradiography (upper panel) or western blotting (lower panel).

 

Figure 8
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  		Fig. 8. RNAi depletion of cyclin B leads to formation of twinned nuclei and blocks lamin depolymerization. (A) Confocal still images from time-lapse recordings of the first zygotic divisions showing cyb-1(RNAi) two-cell-stage (top) or four-cell-stage (bottom) embryos expressing GFP–β-tubulin. The twinned nuclei (arrows) induced upon RNAi depletion of cyclin B exclude soluble GFP-β–tubulin. (B) Confocal still images from time-lapse recordings of the second and third zygotic divisions showing cyb-1(RNAi) embryos expressing YFP-lamin. Localized YFP-lamin staining (arrowheads) remains during mitosis upon RNAi depletion of cyclin B. Bars, 10 µm.

 

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