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Stable kinetochore–microtubule attachments restrict MTOC position and spindle elongation in oocytes

2021; Springer Nature; Volume: 22; Issue: 4 Linguagem: Inglês

10.15252/embr.202051400

1469-3178

Aurélien Courtois, Shuhei Yoshida, Osamu Takenouchi, Kohei Asai, Tomoya S. Kitajima,

Reproductive Biology and Fertility

Report3 March 2021Open Access Source DataTransparent process Stable kinetochore–microtubule attachments restrict MTOC position and spindle elongation in oocytes Aurélien Courtois orcid.org/0000-0001-5988-8794 Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Search for more papers by this author Shuhei Yoshida orcid.org/0000-0002-5640-6128 Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Search for more papers by this author Osamu Takenouchi Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Search for more papers by this author Kohei Asai Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Tomoya S Kitajima Corresponding Author [email protected] orcid.org/0000-0002-6486-7143 Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Aurélien Courtois orcid.org/0000-0001-5988-8794 Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Search for more papers by this author Shuhei Yoshida orcid.org/0000-0002-5640-6128 Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Search for more papers by this author Osamu Takenouchi Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Search for more papers by this author Kohei Asai Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Tomoya S Kitajima Corresponding Author [email protected] orcid.org/0000-0002-6486-7143 Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Author Information Aurélien Courtois1, Shuhei Yoshida1, Osamu Takenouchi1, Kohei Asai1,2 and Tomoya S Kitajima *,1,2 1Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan 2Graduate School of Biostudies, Kyoto University, Kyoto, Japan *Corresponding author. Tel: +81 78 306 3308; E-mail: [email protected] EMBO Rep (2021)22:e51400https://doi.org/10.15252/embr.202051400 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball-like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore–microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore–microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore–microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles. These findings suggest a role for stable kinetochore–microtubule attachments in fine-tuning acentrosomal spindle bipolarity. Synopsis Stable kinetochore–microtubule attachments are required for confining MTOC positions at the spindle poles and for limiting spindle elongation during meiosis I in mouse oocytes. Early bipolar-shaped spindles frequently exhibit small MTOCs in their central region. During metaphase, the majority of MTOCs are positioned at the poles of the bipolar-shaped spindle. A small population of MTOCs are found in the central region of the spindle until they are eventually sorted to the poles. Reducing the stability of kinetochore–microtubule attachments with a phospho-mimetic mutant of Ndc80 leads to defects in restricting MTOCs at the spindle poles and in terminating spindle elongation. Stable kinetochore fibers (K-fibers) are directly connected to MTOCs and to spindle poles. Introduction Spindle bipolarity is a prerequisite for chromosome segregation. In animal somatic cells, two centrosomes act as major microtubule nucleation sites and provide a spatial cue for bipolar spindle formation. In contrast, in acentrosomal cells, such as oocytes, a bipolar spindle forms without canonical centrosomes (Bennabi et al, 2016; Dumont and Desai, 2012; Howe and FitzHarris, 2013; Mogessie et al, 2018; Ohkura, 2015; Radford et al, 2017; Reber and Hyman, 2015). In mouse oocytes, acentriolar microtubule organizing centers (MTOCs) functionally replace centrosomes and serve as major microtubule nucleation sites (Blerkom, 1991; Clift and Schuh, 2015; Maro et al, 1985; Schuh and Ellenberg, 2007). The cytoplasm of oocytes initially carries many MTOCs, which are relocated around chromosomes upon nuclear envelope breakdown (NEBD). Microtubule nucleation from MTOCs leads to the assembly of an apolar microtubule-based structure called a microtubule ball, which then elongates into a bipolar-shaped spindle. Concomitant with the spindle elongation, MTOCs are sorted and relocated into the forming spindle poles (Clift and Schuh, 2015; Schuh and Ellenberg, 2007). These processes establish a bipolar-shaped spindle carrying MTOCs at its two poles. Spindle elongation and MTOC sorting, two characteristic processes involved in spindle bipolarization, require the concerted action of microtubule regulators (Letort et al, 2019), including the plus-end-directed microtubule motor Kif11 (Mailhes et al, 2004; Schuh and Ellenberg, 2007), the microtubule bundle stabilizer HURP (Breuer et al, 2010), the spindle pole-focusing factor NuMA (Kolano et al, 2012), the minus end-directed microtubule motor HSET (Bennabi et al, 2018), the intra-spindle microtubule assembly factor augmin (Watanabe et al, 2016), and the Ndc80 complex (which recruits the antiparallel microtubule crosslinker Prc1 to kinetochores in an oocyte-specific manner) (Yoshida et al, 2020). Despite increasing knowledge about the molecular mechanisms involved in establishing spindle bipolarity, how spindle elongation and MTOC sorting are precisely controlled remains unclear. The mechanism of spindle bipolarity is important to understand as its instability is a hallmark of error-prone human oocytes (Haverfield et al, 2017; Holubcová et al, 2015). Kinetochore–microtubule attachment contributes to the integrity of spindle bipolarity. In centrosomal mitotic cells, the disruption of kinetochore–microtubule attachments can perturb spindle bipolarity, which is pronounced when centrosomal functions are impaired (Lončarek et al, 2007; Moutinho-Pereira et al, 2013; O'Connell et al, 2009; Toso et al, 2009). In mouse oocytes, mutations and knockdowns that perturb kinetochore–microtubule attachment are often associated with spindle bipolarity defects (Gui and Homer, 2013; Sun et al, 2010, 2011; Woods et al, 1999). However, the precise manipulation of the stability of kinetochore–microtubule attachments has not been comprehensively tested. Unlike somatic cells, where sister kinetochore biorientation and entry to metaphase abruptly stabilize kinetochore–microtubule attachments, in oocytes, the stabilization of kinetochore–microtubule attachment is gradual throughout prometaphase and metaphase (Brunet et al, 1999; Davydenko et al, 2013; Kitajima et al, 2011; Yoshida et al, 2015). How the stabilization of kinetochore–microtubule attachment affects the dynamics of spindle elongation and MTOC sorting has not been quantitatively analyzed. In this study, we show that the stability of kinetochore–microtubule attachments is required to confine MTOCs at spindle poles and to limit spindle elongation. Our quantitative analysis demonstrates that MTOC sorting is a gradual process. During metaphase, while the majority of MTOCs are positioned at the poles of the bipolar-shaped spindle, a small population of MTOCs are found in the central region of the spindle until they are eventually sorted to the poles. Reducing the stability of kinetochore–microtubule attachments with a phospho-mimetic form of Ndc80, a major microtubule-anchoring protein at kinetochores, causes defects in restricting MTOCs at the spindle poles and in terminating spindle elongation. Stable kinetochore fibers (K-fibers) are directly connected to MTOCs and to spindle poles. These results suggest the role for stable kinetochore–microtubule attachments in restricting MTOC position and in limiting spindle elongation. Result and discussion MTOC sorting is gradual and frequently leaves small MTOCs in the central region of the bipolar-shaped spindle To investigate acentrosomal spindle assembly during meiosis I, we used high-resolution imaging of MTOCs in mouse oocytes. To visualize the dynamics of MTOCs, we tagged the MTOC marker Cep192 (Clift and Schuh, 2015) with mNeonGreen (mNG-Cep192). We introduced RNAs encoding mNG-Cep192 and the chromosome marker H2B-mCherry into mouse oocytes at the germinal vesicle (GV, prophase I-like) stage and induced meiotic maturation in vitro. The dynamics of MTOCs and chromosomes were recorded with a confocal microscope throughout meiosis I (Movie EV1). We reconstructed the datasets into 3D and manually determined the axis of the spindle at every time point. Side views of the spindle were used to visualize the distribution of MTOCs and chromosomes along the forming spindle axis (Fig 1A). Quantitative analysis showed that the MTOCs gradually moved toward the poles of the bipolarizing spindle until 6 h after NEBD, which coincided with the gradual chromosome congression toward the spindle equator (Fig 1B–D). These observations are consistent with previous reports (Breuer et al, 2010; Clift and Schuh, 2015). We found that even in bipolar-shaped spindles during metaphase (4–6 h after NEBD; Figs 1E and EV1A), a small population of MTOCs were frequently positioned in the middle region of the spindle (Fig 1A and B). These "central MTOCs" were observed in 79% of oocytes at early metaphase (4 h after NEBD, n = 21) and in 47% of oocytes at mid-metaphase (6 h, n = 21). Central MTOCs were observed with another MTOC marker mEGFP-Cdk5rap2 (Fig EV1B). Immunostaining of the MTOC marker pericentrin confirmed the presence of central MTOCs during metaphase (Fig 1F). These observations indicate that MTOC sorting is gradual and continues even in the bipolar-shaped spindle during metaphase. Figure 1. Establishment of 4D quantitative analysis for MTOC dynamics Live imaging of MTOC dynamics during meiosis I. Z-projection images (top) show MTOCs (mNG-Cep192, green) and chromosomes (H2B-mCherry, magenta). Central MTOCs are magnified (squares). 3D-reconstructed spindles are aligned with the spindle axis (middle). Central MTOCs are indicated with arrowheads. Time in h:mm. The kymograph (bottom) shows projected signals on the spindle axis for all time points. Density maps of MTOCs (left) and chromosomes (right) along the spindle axis in a representative oocyte. The color represents the percentage of MTOCs or chromosome volumes, respectively, coded from dark (0%) to white (50% or more of the total volume). Average density maps of 21 oocytes mapped as in (B). Data from six independent experiments are used. Dynamics of MTOC sorting and chromosome congression. Ratio of MTOCs (left) or chromosomes (right) in the polar region versus those in the middle region of the spindle are plotted over time. Dynamics of spindle elongation. The spindle was visualized with EGFP-Map4 (Fig EV1). The length/width ratio of the spindle was measured after 3D reconstruction (n = 13 oocytes from three independent experiments). Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle. Oocytes were fixed and stained for MTOCs (pericentrin, green), chromosomes (Hoechst33342, magenta), and microtubules (α-tubulin, gray). n = 21, 28, 24 oocytes. A representative oocyte fixed at 5 h after NEBD is shown. Arrowheads indicate central MTOCs. Data information: Time after NEBD. Mean ± SD are shown. Scale bars, 10 μm. See also Movie EV1. Source data are available online for this figure. Source Data for Figure 1 [embr202051400-sup-0011-SDataFig1.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Central MTOCs in the bipolar-shaped spindle Live imaging of oocytes expressing EGFP-Map4 (microtubules, green) and H2B-mCherry (chromosomes, magenta). Live imaging of oocytes expressing mEGFP-Cdk5rap2 (microtubules, green) and H2B-mCherry (chromosomes, magenta). Arrowheads indicate central MTOCs. The frequency of oocytes carrying central MTOCs is shown (n = 21 oocytes). Data information: Scale bars, 10 μm. Source data are available online for this figure. Download figure Download PowerPoint A majority of central MTOCs originate from polar regions of the forming spindle To investigate the origin of central MTOCs, we recorded 4D datasets at a higher temporal resolution to track the dynamics of individual MTOCs (Fig 2A). We selected central MTOCs that were positioned in the middle of the spindle later than 4 h after NEBD and retrospectively analyzed their trajectories during prometaphase (Fig 2B). This analysis revealed that a majority of central MTOCs originated from the poles of the bipolar spindle (n = 21/32, Fig 2C and D). These MTOCs spent substantial periods of time in the middle region of the spindle (an average of 69 min and a maximum of 210 min; n = 21; Fig 2C, Movie EV2) and then relocated either to the original (n = 13/21) or to the opposite pole (n = 8/21, Fig 2E and F). We found that a substantial number of the central MTOCs exhibited correlated motions with a closely positioned kinetochore (< 2 μm) for considerable periods of time up to 60.5 min (Fig EV2-EV5, Movies EV3 and EV4), suggesting that central MTOCs can attach to kinetochores. Consistent with this idea, we observed microtubules linking central MTOCs to a closely positioned kinetochore at early metaphase (Fig EV3A). Close positioning of central MTOCs did not significantly affect the stability of kinetochore–microtubule attachments (Fig EV3B) and the levels of phosphorylated Ser55 of Ndc80 (Fig EV3C), a phosphorylation that destabilizes kinetochore–microtubule attachment (Cheeseman et al, 2006; DeLuca et al, 2006). After showing correlated motions with a kinetochore, the MTOCs eventually moved to the original pole or the opposite pole (Fig EV2-EV5, Movies EV3 and EV4). These results suggest that central MTOCs can undergo a dynamic cycle of attachment and detachment to kinetochores. These observations indicate that MTOC sorting is highly asynchronous and includes active exchanges of MTOCs between the two forming spindle poles. Figure 2. Central MTOCs originate from spindle poles Live imaging of MTOC and kinetochore dynamics. MTOCs (mNG-Cep192, green) and kinetochores (tdTomato-CENP-C, magenta) in B6D2F1 oocytes are shown. Time in h:mm after NEBD. Circles indicate central MTOCs, which are positioned in the middle region of the spindle later than 4 h after NEBD. Arrows indicate displacement of the MTOCs. Scale bar, 10 μm. Tracking analysis of central MTOCs. In the left graph, the positions of central MTOCs in (A) along the spindle axis are shown over time. MTOC positions were used to calculate distance to the spindle equator. In the right graph, lines showing temporal changes in MTOC-equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle. As in (B), the trajectories of central MTOCs (n = 25 MTOCs of 8 oocytes from three independent experiments) were analyzed and their temporal changes in MTOC–equator distance are shown (Left graph). Central MTOCs were categorized into two groups: (1) ones that came from polar regions of the spindle (n = 21, middle graph) and (2) others that stayed in the middle region throughout the period before reaching a position closest to the equator (n = 4, right graph). Origin of central MTOCs. Origins were categorized based on the results shown in (C). Note that 7 central MTOCs came from outside of the spindle and are not included in (C). Destination of central MTOCs. The tracks of central MTOCs that originated from polar regions were used (n = 21 MTOCs). The pole of origin was defined as Pole 1, while the other pole was defined as Pole 2. MTOC positions along the spindle axis (positive values for those closer to Pole 1) over time are shown. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. Note that central MTOCs moved back to the original pole (Pole 1) or switched their positions to the opposite pole (Pole 2). Destination of central MTOCs that originated from a polar region was categorized as in (E). Data information: See also Movie EV2. Source data are available online for this figure. Source Data for Figure 2 [embr202051400-sup-0012-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Central MTOCs can be positioned close to kinetochores Live imaging of MTOC and kinetochore dynamics. Z-projected and 3D-reconstructed images for MTOCs (mNG-Cep192, green) and kinetochores (tdTomato-CENP-C, magenta) are shown. The 3D-reconstructed images are aligned based on the spindle axis, with highlights on an MTOC and a pair of kinetochores. Time in h:mm after NEBD. Scale bar, 10 μm. Cyan circles follow an MTOC in the middle of the spindle. Magenta circles follow a kinetochore that transiently positions close to the MTOC. Tracking of MTOCs and kinetochores. The positions of MTOCs (cyan) and kinetochores (magenta), which are highlighted in (A), along the spindle axis are shown over time. Time after NEBD. Dotted boxes represent periods when the kinetochore-MTOC distance was < 2 μm. Another example of the image dataset acquired from the experiment shown in (A). Images are presented as in (A). The dataset shown in (C) was analyzed and presented as in (B). Close positioning of MTOCs and kinetochores is frequently observed. Lines indicate periods when oocytes exhibited close positioning of MTOCs and kinetochores (< 2 μm). We categorized kinetochore-proximal MTOCs into three groups: (1) MTOCs that originated from a pole, positioned close to a kinetochore, and then moved back to the original spindle pole (green), (2) MTOCs that originated from a pole, positioned close to a kinetochore, and then moved to the opposite spindle pole (blue) and, (3) MTOCs that did not originate from spindle poles and are positioned close to the kinetochore (black). Data information: See also Movies EV3 and EV4. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Relationship between central MTOCs and kinetochores Central MTOCs can attach to kinetochores. Oocytes at 4 h and 5 h after NEBD were treated with a cold buffer for 5 min before fixation. Cold-stable microtubules were visualized by immunostaining oocytes for microtubules (α-tubulin, green), kinetochores (ACA, magenta), pericentrin (gray) and chromosomes (Hoechst33342, blue). Z-projection images and magnified z-slices are shown. The frequency of oocytes containing central MTOC–kinetochore attachment and/or overlap is shown (n = 19, 18 oocytes from three independent experiments). Scale bar, 10 μm. Close positioning of central MTOCs does not affect stable kinetochore–microtubule attachment. Kinetochores were categorized into two groups: (1) ones that were positioned close to central MTOCs (< 1 μm) and (2) others. The frequency of stable end-on microtubule attachment on each category is shown (n = 12, 6 oocytes from three independent experiments). Mean ± SD are shown. n.s., not significant by two-tailed Student's t-test. Close positioning of central MTOCs does not affect phosphorylated Ser55 of Ndc80. Kinetochores were categorized into two groups: (1) ones that positioned close to central MTOCs (< 1 μm) and (2) others. Relative intensities of phospho-Ser55 to ACA signals were calculated (n = 266, 14 kinetochores of 7 oocytes from three independent experiments). Mean ± SD are shown. n.s., not significant by two-tailed Student's t-test. Scale bar, 10 μm. Temporal correlation of MTOC sorting with chromosome alignment and anaphase onset. Oocytes expressing mNG-Cep192 and H2B-SNAP labeled with SNAP-Cell 647-SiR or H2B-mCherry were monitored by live imaging (n = 27 from seven independent experiments). The timings of the removal of all central MTOCs, chromosome alignment, and anaphase onset were determined. Correlation coefficients are shown. Time after NEBD (h). Close positioning of central MTOCs and chromosomes does not predict chromosome segregation errors. Oocytes expressing H2B-SNAP labeled with SNAP-Cell 647-SiR (chromosomes), tdTomato-CENP-C (kinetochores), and mNG-Cep192 (MTOCs) were cultured in the presence of 60 nM nocodazole and monitored by live imaging. Images were reconstructed in 3D. MTOCs are shown in green. A central MTOC that transiently positioned close to a chromosome is shown in magenta. This chromosome (shown in red) underwent normal segregation at anaphase. Chromosomes that underwent segregation errors are shown in blue. Other chromosomes are shown in gray. Kinetochore positions are marked with orange spheres. The frequency of segregation errors are shown. All chromosomes (n = 760 chromosomes of 38 oocytes) and chromosomes that positioned close to central MTOCs (n = 9 from 8 oocytes). The unit of the grid is 5 μm. Time after NEBD (h:mm). See also Movie EV5. Central MTOCs do not predict chromosome segregation errors in aged oocytes. Oocytes were collected from young (2-month-old) and naturally aged mice (17–22 months old). Oocytes expressing mNG-Cep192 and H2B-mCherry were monitored by live imaging. The number of central MTOCs was determined at 4 and 5 h after NEBD (n = 40, 22 from three independent experiments). Boxes and whiskers show 25–75 and 10–90 percentiles, respectively. Images for a representative aged oocyte exhibiting prematurely separated chromosomes (white arrowhead) are shown. Yellow arrowheads indicate central MTOCs. Time after NEBD (h:mm). Scale bar, 10 μm. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Ndc80 phospho-mutants modify kinetochore–microtubule attachment stability Schematic representation of kinetochore–microtubule attachment. Phosphorylation levels of the Ndc80 complex at the kinetochore are a determinant of the stability of kinetochore (KT)-microtubule (MT) attachments. High phosphorylation levels allow unstable attachments such as lateral attachments. Low phosphorylation levels promote stable attachments such as end-on attachments. Phospho-mimetic and phospho-deficient forms of Ndc80 (S4, S5, T8, S15, S44, T49, S55, S62, and S68 were substituted to aspartic acid "Ndc80-9D" or alanine "Ndc80-9A", respectively) can be used as tools to manipulate the stability of kinetochore–microtubule attachments. Ndc80 phospho-mutants modify kinetochore–microtubule attachment stability. Ndc80f/f Zp3-Cre oocytes were collected and microinjected with RNAs of wild-type (Ndc80-WT), phospho-mimetic (Ndc80-9D) or phospho-deficient (Ndc80-9A) forms of Ndc80. Cold-stable microtubules were visualized by immunostaining oocytes 7 hours after NEBD for microtubules (α-tubulin, cyan), kinetochores (ACA, orange), and chromosomes (Hoechst33342, magenta). Z-projection images are shown. Magnified images of kinetochores are categorized: end-on attachments (dark blue box), lateral attachments (light blue box), and unattached (dot-lined box). Scale bar, 10 μm. Percentages of kinetochore–microtubule attachments in (B). All kinetochores were analyzed (n = 200 kinetochores of 5 oocytes from two independent experiments). Anaphase entry timing. Ndc80f/f (Ndc80-intact) oocytes are used as a control. Ndc80f/f Zp3-Cre (Ndc80-deleted) oocytes expressing Ndc80-WT, Ndc80-9D, or Ndc80-9A were monitored with the chromosome marker H2B-mCherry (n = 15, 25, 25, 42, and 55 oocytes, respectively, from at least 3 independent experiments). Time after NEBD. Note that Ndc80f/f Zp3-Cre oocytes expressing no Ndc80 construct exhibited an accelerated anaphase onset that is consistent with defects in the spindle checkpoint. Ndc80-9D-expressing oocytes did not undergo anaphase, consistent with spindle checkpoint activation by unstable kinetochore–microtubule attachments. Ndc80-9A-expressing oocytes tended to exhibit an earlier anaphase onset, in agreement with accelerated checkpoint satisfaction by stable kinetochore–microtubule attachments. Boxes show 25–75 percentiles and whiskers encompass data points within 1.5 times the interquartile range. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Kinetochore–microtubule attachment stability is not absolutely required for bipolar microtubule organization Ndc80-9D-expressing oocytes do not exhibit increased collapse in spindle shape. Microtubules (EGFP-Map4) and chromosomes (H2B-mCherry) were visualized in Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, Ndc80-9D, or Ndc80-9A. The aspect ratio (length/width) of 3D-reconstructed spindles was measured over time (n = 18, 18, and 18 oocytes, respectively, from three independent experiments; identical to the dataset shown in Fig 4). Experimental pipeline for the quantification of microtubule organization. Oocytes were imaged with the microtubule plus-end marker EB3-3mEGFP (green) and the chromosome marker H2B-mCherry (magenta) at 5 h after NEBD. The comets of EB3-labeled microtubule plus-ends were detected following image processing for peak enhancement and background subtraction. The comets were tracked to visualize their movements. The spindle was divided into 21 equal regions between the two spindle poles along the spindle axis, and divisions 4–6 and 16–18 were defined as polar regions. In the polar regions, comets moving toward the inside of the spindle were categorized as inward comets (magenta lines), and the others as outward comets (green lines). Inward comets were predominant in the polar regions of the spindle, which represents bipolar microtubule organization. EB3 dynamics are largely unaffected by kinetochore–microtubule stability. Zp3-Cre Ndc80f/f oocytes expressing Ndc80-WT, Ndc80-9D, or Ndc80-9A were analyzed as in (B). EB3 dynamics are not significantly affected. Total number of comets going inwards and outwards in the polar regions of the spindle (n = 14, 13, 16 oocytes from at least two independent experiments) is shown. Welch's t-test was performed. Boxes show 25–75 percentiles, and whiskers encompass data points within 1.5 times the interquartile range. Data information: Scale bars, 10 μm. See also Movie EV9. Source data are available online for this figure. Download figure Download PowerPoint Central MTOCs do not predict chromosome segregation errors These observations led us to investigate potential links between central MTOCs and chromosome segregation errors in oocytes. The time when the last central MTOC moved to a pole was correlated with the completion of chromosome alignment and with the onset of anaphase (Fig EV3D), consistent with the idea that central MTOCs pose a problem for chromosome segregation. However, we observed no chromosome segregation errors under our imaging condition (0/16 oocytes), which indicated that central MTOCs did not cause chromosome segregation errors in normal cultures. To artificially induce chromosome segregation errors, we treated oocytes with low-dose (60 nM) nocodazole, a microtubule depolymerizer. Chromosome tracking analysis showed that ~12% of all chromosomes underwent segregation errors (90/760 chromosomes from 38 oocytes; Fig EV3E and Movie EV5). In this dataset, we detected 9 chromosomes that transiently exhibited correlated motions with a closely positioned kinetochore during metaphase. Only 1 of such chromosomes underwent chromosome segregation error (1/9 chromosomes, ~11%; Fig EV3E). Thus, under this experimental condition, central MTOCs positioned close to chromosomes did not predict chromosome segregation errors. Chromosome segregation errors can also be induced by maternal aging. We found no significant increase in the frequency of the appearance of central MTOCs in naturally aged (17–22-month-old)