Fig 1: Ndc80 is essential for spindle bipolarization in MI but not in MII.a Live imaging of Ndc80f/f (control) and Ndc80f/f Zp3-Cre (Ndc80-deleted) oocytes expressing EGFP-Map4 (microtubules, green) and H2B-mCherry (chromosomes, magenta). Spindle shapes were reconstructed in 3D. Asterisks show chromosomes that fell off the spindle. Anaphase I was defined based on characteristic chromosome and spindle dynamics (see Methods). Three independent experiments were performed. See also Supplementary Movie 1, 2, and 3. b Establishment of spindle bipolarity requires Ndc80 in MI. Spindle shapes in 3D were categorized based on the aspect ratio, surface irregularity, and stability (see Methods). Spindles that maintained a bipolar state for 1 h or longer with no collapse prior to anaphase I onset were categorized as stable bipolar spindles. c Ndc80 is required for preventing aneuploidy in eggs. The number of chromosomes were counted at metaphase II (n = 28, 28 oocytes from three independent experiments). Bars represent mean. See also Supplementary Movie 4. d The Ndc80 complex and Ndc80?N/Nuf2?N construct. In the Ndc80 complex, Ndc80 and Nuf2 directly bind to microtubules via the head domains at their N-termini, and to Spc24 and Spc25 at their C-termini. Ndc80 contains a loop domain in the central region, which recruits microtubule-binding proteins. Ndc80?N (a.a. 461–642) and Nuf2?N (a.a. 276–463) contain neither the head nor loop domain but retain the Spc24- and Spc25-binding domains. e Ndc80?N/Nuf2?N rescues spindle defects. Ndc80f/f Zp3-Cre oocytes coexpressing Ndc80?N and Nuf2?N (Ndc80?N/Nuf2?N) and those expressing full-length Ndc80 (Ndc80-WT) were monitored for spindle formation. EGFP-Map4 (microtubules, green) and H2B-mCherry (chromosomes, magenta) signals at metaphase I (5.5 h after NEBD) are shown. Spindle shapes were reconstructed in 3D at metaphase I (5.5 h after NEBD) and categorized based on the aspect ratio and surface irregularity (n = 23, 23, 21 oocytes from three independent experiments). Mean +/- SD are presented. **p = 0.0026; ***p = 0.0002; by two-tailed unpaired Student’s t-test. See also Supplementary Movie 5. Scale bars, 10 µm.
Fig 2: Ndc80/Nuf2 concentrates dynamic Prc1 at kinetochores for spindle bipolarization.a Dynamic exchange of Prc1 at kinetochores. In oocytes at prometaphase I (1.5 h after NEBD), SunTag-Prc1 (24xGCN4-Prc1 coexpressed with scFv-sfGFP, green) signals at kinetochores were bleached, and the recovery was monitored (n = 5, 5, 5 kinetochores). Note that the recovery curve of SunTag-Prc1 signals indicates the turnover of Prc1 at kinetochores rather than the turnover of scFv on 24xGCN4 in this time range, which was confirmed by Ndc80-SunTag (Ndc80-24xGCN4 coexpressed with scFv-sfGFP) exhibiting similar recovery curves to Ndc80-sfGFP. See also Supplementary Movie 7. b Kif11-dependent Prc1 enrichment along kinetochore-proximal microtubules of the bipolar spindle. Control or monastrol-treated oocytes at metaphase I (4–6 h after NEBD) were stained for Prc1 (green), microtubules (magenta), and DNA (Hoechst33342, blue). The oocytes were treated with a cold buffer for 1 min before fixation to facilitate antibody penetration into the spindle. Prc1 signals along kinetochore-proximal microtubule bundles were measured, and their ratio to microtubule signals was calculated (n = 25, 25 locations from 5, 5 oocytes. Three independent experiments were performed). ****p < 0.0001 (p = 8.1E-07) by two-tailed unpaired Student’s t-test. c Prc1 overexpression rescues spindle defects in Ndc80-deleted oocytes. Ndc80f/f Zp3-Cre oocytes overexpressing mEGFP-HURP, mEGFP-Kif11, mNeonGreen-HSET, or mNeonGreen-Prc1 were immunostained at metaphase I (5.5 h after NEBD). Spindle shapes were reconstructed in 3D and categorized (n = 29, 18, 18, 18, 24 oocytes from three independent experiments). ****p < 0.0001 (p = 5.1E-05) by two-tailed unpaired Student’s t-test. n.s., not significant. Scale bars, 10 µm. Mean +/- SD are presented in a–c.
Fig 3: Relationship between miR-344d-3-5p and Prc1 (and Nuf2).A, B Relative luciferase activities of wild type (WT) and mutated (MUT) Prc1 reporter gene vectors in B16 cells co-transfected with miR-344d-3-5p mimic/miR-344d-3-5p mut. C, D Relative luciferase activities of wild-type (WT) and mutated (MUT) Nuf2 reporter gene vectors in B16 cells co-transfected with miR-344d-3-5p mimics/miR-344d-3-5p mut. E PRC1 protein expression following transfection with miR-344d-3-5p mimics/miR-344d-3-5p mut. F Quantitative analysis of the protein level of PRC1 in miR-344d-3-5p mimics/miR-344d-3-5p mut-transfected B16 cells. G NUF2 protein expression when transfected with miR-344d-3-5p mimics/miR-344d-3-5p mut. H Quantitative analysis of the protein level of NUF2 in miR-344d-3-5p mimics/miR-344d-3-5p mut-transfected B16 cells. I Quantitative analysis of the mRNA level of Prc1 in B16 cells after treated with ATRA and PB-4 by RT-qPCR. J PRC1 protein expression in B16 cells after treatment with ATRA and PB-4. K Quantitative analysis of the protein level of PRC1 in B16 cells following treatment with ATRA and PB-4. L Quantitative analysis of the mRNA level of Nuf2 in B16 cells after treatment with ATRA and PB-4 by RT-qPCR. M NUF2 protein expression in B16 cells after treatment with ATRA and PB-4. N Quantitative analysis of the protein level of NUF2 in B16 cells after treatment with ATRA and PB-4. n = 3. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01 compared with the corresponding control group by Student’s t-test.
Fig 4: Effect of silencing lncRNA-Gm31932 on PRC1- and NUF2-associated pathways.A Quantitative analysis of the mRNA level of Prc1 in B16 cells after silencing lncRNA-Gm31932 by RT-qPCR. B PRC1 protein expression in B16 cells after silencing lncRNA-Gm31932. C Quantitative analysis of the protein level of PRC1 after silencing lncRNA-Gm31932. D Quantitative analysis of the mRNA level of Nuf2 in B16 cells after silencing lncRNA-Gm31932 by RT-qPCR. E NUF2 protein expression in B16 cells after silencing lncRNA-Gm31932. F Quantitative analysis of the protein level of NUF2 after silencing lncRNA-Gm31932. G Western blotting for CDK2, CDC2, and Cyclin B1 in B16 cells after transfection with siRNA-Gm31932-174. H Quantitative analysis of the protein levels of CDK2, CDC2, and Cyclin B1 in B16 cells after transfection with siRNA-Gm31932-174. I Western blotting of P21 and P27 in B16 cells after transfection with siRNA-Gm31932-174. J Quantitative analysis of the protein level of P21 and P27 in B16 cells after transfection with siRNA-Gm31932-174. K Western blotting of WNT1 and ß-catenin in B16 cells after transfection with siRNA-Gm31932. L Quantitative analysis of the protein level of WNT1 and ß-catenin in B16 cells after transfection with siRNA-Gm31932. n = 3. Data are presented as the mean ± SD. **P < 0.01 compared with the corresponding control group by one-way ANOVA followed by the Dunnett correction.
Fig 5: Ndc80/Nuf2 promotes spindle bipolarization by recruiting Prc1 at kinetochores.a Ndc80/Nuf2 recruits Prc1 at kinetochores. Ndc80f/f (control) and Ndc80f/f Zp3-Cre (Ndc80-deleted) oocytes expressing full-length Ndc80 (Ndc80-WT) or Ndc80?N/Nuf2?N were immunostained for Prc1 (green), kinetochores (ACA, magenta), and DNA (Hoechst33342, blue). Kinetochores are magnified. In the plot, spots and squares correspond to individual kinetochores and oocytes, respectively (n = 200, 198, 200, 200 kinetochores from 5, 5, 5, 5 oocytes). ****p < 0.0001 (exact values are shown in the panel) by two-tailed unpaired Student’s t-test. b Ndc80-bound beads associate with Prc1. Oocytes expressing mEGFP or Ndc80-mEGFP (green) were microinjected with anti-GFP beads, fixed at prometaphase I, and immunostained for Prc1 (magenta) (see Methods). In the plot, spots correspond to individual beads (n = 50, 48 beads from 5, 5 oocytes). ****p < 0.0001 (p = 4.7E-87) by two-tailed unpaired Student’s t-test. c Ndc80/Nuf2–Prc1 acts at kinetochores. Ndc80f/f Zp3-Cre oocytes expressing Ndc80?N-4A/Nuf2?N (Y564A, Q565A, L566A, and T567A mutations; indicated as ‘NN?N-4A’) or its Spc-fused form Ndc80?N-4A-Spc25C/Nuf2?N-Spc24C (indicated as ‘NN?N-4A tethered at KTs’) were monitored. Spc25C (a.a. 120–226) and Spc24C (a.a. 122–201) are kinetochore-targeting domains. EGFP-Map4 (microtubules, green) and H2B-mCherry (chromosomes, magenta) at metaphase I (5.5 h after NEBD) are shown. Spindles were reconstructed in 3D and categorized based on the aspect ratio and surface irregularity. The frequency of oocytes that exhibited a bipolar-shaped spindle is shown (n = 19, 18 oocytes from three independent experiments). ***p = 0.0004 by two-tailed unpaired Student’s t-test. d Ndc80?N/Nuf2?N requires Prc1 to promote spindle bipolarization. Prc1 was depleted by RNAi in Ndc80f/f Zp3-Cre oocytes, and Ndc80?N/Nuf2?N was expressed. EGFP-Map4 (microtubules, green) and H2B-mCherry (chromosomes, magenta) at metaphase I (8 h after NEBD) are shown. Spindle shapes were reconstructed in 3D and categorized based on the aspect ratio and surface irregularity. The frequency of oocytes that exhibited a bipolar-shaped spindle is shown (n = 27, 27 oocytes from three independent experiments). **p = 0.0021 by two-tailed unpaired Student’s t-test. e Summary of Ndc80/Nuf2 constructs and their capacities to rescue spindle bipolarization. Scale bars, 10 µm. Mean +/- SD are presented in a–d.
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