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. 2020 May 27;11:2652. doi: 10.1038/s41467-020-16488-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Ndc80/Nuf2 recruits Prc1 at kinetochores. Ndc80^f/f (control) and Ndc80^f/f Zp3-Cre (Ndc80-deleted) oocytes expressing full-length Ndc80 (Ndc80-WT) or Ndc80ΔN/Nuf2ΔN 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. Ndc80^f/f Zp3-Cre oocytes expressing Ndc80ΔN-4A/Nuf2ΔN (Y564A, Q565A, L566A, and T567A mutations; indicated as ‘NNΔN-4A’) or its Spc-fused form Ndc80ΔN-4A-Spc25C/Nuf2ΔN-Spc24C (indicated as ‘NNΔN-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 Ndc80ΔN/Nuf2ΔN requires Prc1 to promote spindle bipolarization. Prc1 was depleted by RNAi in Ndc80^f/f Zp3-Cre oocytes, and Ndc80ΔN/Nuf2ΔN 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.