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Cyclins B1 and B2 are subtypes of cyclin B, a regulatory subunit of a maturation/M-phase promoting factor, and they are also highly conserved in many vertebrate species. Cyclin B1 is essential for mitosis, whereas cyclin B2 is regarded as dispensable. However, the overexpression of the cyclin B2 N-terminus containing the cytoplasmic retention signal, but not cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes and embryos. Here we show that endogenous cyclin B2 was localized in and around the germinal vesicle. The perinuclear localization of cyclin B2 was perturbed by the overexpression of its N-terminus containing the cytoplasmic retention signal, which resulted in a spindle defect. This spindle defect was rescued by the overexpression of bipolar kinesin Eg5, which is located at the perinuclear region in the proximity of endogenous cyclin B2. These results demonstrate that the proper localization of cyclin B2 is essential for bipolar spindle formation in Xenopus oocytes.
Fig. 1.
Distribution of Myc-tagged cyclin B2 N-terminus. Immature oocytes expressing B2D (A) or B2DC (B) are stained with anti-Myc antibodies. Immunostaining of uninjected oocytes as a control is shown (C). A higher magnified image of the perinuclear region of oocytes injected with B2D or B2DC (D and E) mRNA. The Western blot analysis of immature oocytes injected with Myc-tagged B2D or B2DC (F). GV represents germinal vesicle. All Figures are meridional sections, and their animal pole points toward the upside. Scale bars represent 150 μm (A–C) and 50 μm (D and E).
Fig. 2.
The subcellular localization of endogenous cyclin B2 and the effect of the overexpression of the cyclin B2 N-terminus. Xenopus immature oocyte (Oo) and matured egg (Egg) extracts (A), and in vitro-synthesized cyclins B1 and B2 proteins in a reticulocyte lysate (B) were immunoblotted with affinity-purified anti-B2δN antibodies. As a control, the same volume of a reticulocyte lysate to which no mRNA was added was used. Arrowheads represent endogenous cyclin B2. Sizes of protein markers (kDa) are indicated on the left side. Immature uninjected oocytes (C) or oocytes injected with B2DC (D) are stained with anti-B2δN antibodies. A higher magnified image of the perinuclear region of uninjected oocytes is also shown (E). The same perinuclear regions of immature oocytes injected with B2DC (F), B2D (G) or B1DC (H) are stained with anti-B2δN antibodies. GV represents germinal vesicle. All Figures are meridional sections, and their animal pole points toward the upside. Scale bars represent150 μm (A and B) and 50 μm (C–F).
