Dumont J et al. (2007), A centriole- and RanGTP-independent spindle ass...

XB-ART-35182

J Cell Biol 2007 Jan 29;1763:295-305. doi: 10.1083/jcb.200605199.

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A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes.

Dumont J , Petri S , Pellegrin F , Terret ME , Bohnsack MT , Rassinier P , Georget V , Kalab P , Gruss OJ , Verlhac MH .

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Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin beta. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 kpnb1 pbrm1 ran ranbp1 rcc1 tub

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	Figure 1. Spindle formation during mouse meiotic maturation. (A) Time-lapse microscopy of phase contrast (DIC), Hoechst-stained chromosomes (Hoechst), and tubulin-GFP RNA-injected (Tub-GFP) oocytes. Images were taken every 15 min. (B) Time-lapse microscopy of phase contrast (DIC) and tubulin-GFP RNA-injected (Tub-GFP) oocytes. Images were taken every 20 min. Times after GVBD are indicated in the bottom left corner. n = 33. Bars, 10 μm.
	Figure 2. Characterization of Ran and its regulators in mouse and X. laevis oocytes. (A, left) The amount of Ran protein is stable during meiotic maturation of mouse oocytes. 30 immature (GV) or mature (MII) mouse oocytes were collected and immunoblotted using a monoclonal anti- Ran antibody and an anti-tubulin antibody. (right) The amount of RCC1 increases slightly during meiotic maturation of mouse oocytes. 100 immature (GV), 4 h after GVBD (MI), or mature (MII) mouse oocytes were collected and immunoblotted using a polyclonal anti-RCC1 antibody and an anti-tubulin antibody. (B, left) RCC1 levels increase during meiosis in X. laevis oocytes, but RanGAP, RanBP1, and Ran levels remain constant. Oocyte lysates (GV) or egg lysates (MII) were immunoblotted using anti-RanGAP, anti-RCC1, anti-RanBP1, and anti-Ran antibodies. (top right) Histone H1 kinase activity (arbitrary units) during meiotic maturation induced by progesterone. (bottom right) Increase of RCC1 amount during meiosis resumption in X. laevis. Two oocytes were used for each time point.
	Figure 3. Detection of a gradient of RanGTP-induced release of importin β in live mouse oocytes. (A and B) Live MII-arrested mouse oocyte injected with Rango and histone-RFP encoding RNA. (A) Image of phase contrast (DIC) merged with the image of the chromosomes (in green). (B) Pseudocolored IFRET/ICFP ratio of the same oocyte expressing Rango. (C) IFRET/ICFP ratio line scans performed perpendicularly to the metaphase plate from 10 MII oocytes, where the position of the chromosomes was arbitrarily set at 0. (D) Time-lapse microscopy of mouse oocytes injected with histone-RFP and Rango encoding RNA. The top panel corresponds to the phase contrast (DIC) merged with the image of the chromosomes (in green). The bottom panel corresponds to the pseudocolored IFRET/ICFP ratio emitted by the Rango probe. Time after GVBD is indicated in the top left corner on the DIC time-lapse image. The two bottom panels correspond to the kymograph of a cropped region containing the chromosomes from the same time lapse (one image per 30 min) plotted against time. n = 27. BD, GVBD; PB1, first polar body extrusion. (E) The gradient of RanGTP-regulated release of importin β is perturbed by RanT24N or RanQ69L throughout meiotic maturation. Immature (GV; 1, 2, and 3) or metaphase II–arrested (MII; 4, 5, and 6) mouse oocytes injected with Rango and histone-RFP encoding RNA alone (1 and 4; n = 30) or further injected either with RanT24N (2 and 5; n = 41 for GV and 37 for MII) or RanQ69L (3 and 6; n = 43 for GV and 36 for MII). (top) Image of phase contrast (DIC) merged with the image of the chromosomes (in green); (bottom) pseudocolored IFRET/ICFP ratio of the corresponding oocyte expressing Rango. The same scale of colors was applied to all samples. Bars, 20 μm.
	Figure 4. Excess or reduced levels of RanGTP induce spindle defects during meiosis I and II of mouse oocytes. Time-lapse microscopy of phase contrast (DIC) and tubulin-GFP (Tub-GFP) of oocytes injected with tubulin-GFP together with RanWT (A) or RanQ69L (B) or RanT24N (C) RNA. Images were taken every 15 min. Arrowheads indicate asters. Times after GVBD are indicated in the bottom right corner. n = 34 (RanWT), 26 (RanQ69L), and 75 (RanT24N). Bars, 10 μm.
	Figure 5. MI spindle defects induced by excess or reduced levels of RanGTP do not compromise homologous chromosome segregation. (A and B) RanQ69L-injected oocytes assemble longer spindles with poles defects, whereas RanT24N induces a lack of MT assembly associated with a delay in spindle formation. (A) Immature oocytes were microinjected with RanWT, RanQ69, or RanT24N, collected 5 or 7 h after GVBD and immunostained for MTs (green) and DNA (red). Oocytes were analyzed by confocal microscopy, and all confocal sections were projected. Proportions of each phenotype are indicated in the bottom right corner. (B) Statistics of spindle length and width in control and RanQ69L-injected oocytes. Error bars indicate SD. (C) Still images taken from time-lapse microscopy of tubulin-GFP (Tub-GFP) of oocytes injected with tubulin-GFP together with RanQ69L or RanT24N RNA. (D) Meiosis I spindles assembled with modified RanGTP levels can segregate homologous chromosomes. Noninjected control oocytes were collected 6 h (MI) or 12 h (MII) after GVBD and submitted to chromosome spread preparation. MI preparation shows only bivalent chromosomes with chiasmata, whereas MII preparation shows only univalent chromosomes (1 and 2). RanT24N- or RanQ69L-injected oocytes were collected 12 h after GVBD (MII) and similarly submitted to chromosome spread preparation (3 and 4). In both cases, only univalent chromosomes are seen. 25 oocytes were analyzed for each condition. Bars, 10 μm.
	Figure 6. Spindle defects induced by excess or reduced levels of RanGTP during meiosis II. (A) MII oocytes collected 15 h after GVBD and immunostained for MTs (green) and DNA (red). (1) Control noninjected oocyte; (2–6) immature oocytes were microinjected with RanWT (2), RanQ69L (3 and 4), or RanT24N (5 and 6). Oocytes were analyzed by confocal microscopy, and all confocal sections were projected. Bar, 20 μm. (B) Statistics of the experiment described above. “No spindle” indicates oocytes showing monopolar structure or a single aster around chromosomes with or without numerous asters in the cytoplasm. “Bipolar with asters” indicates oocytes showing an opened bipolar structure around chromosomes connected to numerous asters in the cytoplasm, and “bipolar” indicates oocytes showing a normal bipolar MII spindle. The number in parentheses corresponds to the total number of oocytes analyzed. Error bars indicate SD. (C) MII-arrested oocytes were activated with strontium to allow the metaphase II–anaphase II transition. Oocytes were observed 5 h after Sr2+ activation either after chromosome labeling to visualize the pronuclei (7–10) or by transmitted light to visualize the second polar bodies (11–13). RanT24N- injected oocytes presented a 2-h delay in their progression to interphase as indicated by the retarded decondensation of chromatin in these oocytes compared with controls. Immature oocytes were either noninjected (7 and 11) or microinjected with RanWT (8 and 12) or RanT24N (9, 10, and 13) and then activated after their arrest in MII and observed 5 h after activation.
	Figure 7. Down-regulation of RCC1 during meiotic maturation in X. laevis oocytes does not compromise meiosis I spindle formation but affects the meiosis II spindle. (A) Western blot on oocyte lysates using antibodies against RCC1 or PP1G as loading control, before (−pg) and 12 h after induction of maturation with progesterone in nontreated (NI), control injected (Cont as), or RCC1 knockdown (RCC1 as) samples. (B) Histone H1 kinase activities were measured with time after GVBD in control injected (triangles) and RCC1 knockdown (circles) oocytes. (C) Western blot on oocyte lysates using antibodies against RCC1 and Ran as loading control, before and after induction of maturation with progesterone in nontreated controls or antisense RCC1-injected oocytes. Samples were taken before progesterone addition or after GVBD (GVBD corresponds to time 0). (D–G) Representative images (D and F) and quantitative analysis of imaged structures (E and G) of spindles visualized in MI (D and E; 1.5 h after GVBD) or MII (F and G; 12 h after GVBD) by indirect immunofluorescence with antibodies against α-tubulin (red) or Cytox green (green) of oocytes treated as in (A, B, and C). Bars, 20 μm.

References [+] :

Askjaer, Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos. 2002, Pubmed