Fig 2: Computational modelling of chromosome distribution.(a) Cartoon showing a simplified mechanistic model of mitotic spindles with various components and forces. (b) Model of the kMT-KT attachment configuration in control Luc siRNA-treated Cos-7 cells; the model shows that EB1–Ska complex microtubule-binding structures couple kMT with Ndc80 in control cells. (c,d) are simulation snapshot of chromosomal configuration and distribution around the metaphase plate in these cells, respectively. The snapshots of in silico phenotypes show perfectly aligned chromosomes at the metaphase plate. (e–g) are the model of the kMT–KT attachment, simulation snapshot of chromosomal configuration and distribution in EB1-depleted cells, respectively. In the EB1-depleted cells, due to absence of EB1–Ska structures, the depolymerizing kMTs often fail to recover leading to defective kMT–KT attachment. The in silico snapshot shows dispersed chromosomes around the metaphase plate. Note that some residual Ska proteins are still present on the kMT that can weakly interact with Ndc80. (h–j) are the model of the kMT–KT attachment, simulation snapshot of chromosomal configuration and distribution in Ska1-depleted cells, respectively. The snapshot shows dispersed chromosomes with wider distribution around the metaphase plate. Some dispersed oligomeric structures of EB1 present on the microtubule are able to weakly interact with Ndc80. (k–m) correspond to the model, chromosomal configuration and distribution when EB1 and Ska1 are co-depleted. The snapshot and the distribution plot show chromosomes scattered all over the spindles. Simulation was carried out with ∼100 samples in each case.

Fig 3: The Centromeric CENP-A Domain Decompacts as Oocytes Age and MII Kinetochores Built upon It Fragment into Lobes(A) Centromeric CENP-A fluorescence intensity (integrated density of the centromeric region of interest [ROI] − mean background fluorescence × ROI) in 42 mouse MII eggs (3 independent experiments). 100% was assigned to the mean intensity of young groups. Box plots show median (horizontal white lines), mean (small white squares), 25th and 75th percentiles (boxes), and 5th and 95th percentiles (whiskers).(B) Representative metaphase-II chromosomes from a young and an old mouse egg (8- and 62-week-old females, respectively) visualized with Airyscan microscopy (z projections of 14–16 sections, acquired every 0.18 μm). Centromeres (green, CENP-A) and DNA (blue, Hoechst) are shown. Scale bars represent 2 μm in overviews, and 0.5 μm in insets.(C) Quantification of MII centromere circularity across the two age groups. A circularity value of 1.0 indicates a perfect circle. 1,776 measurements from 14 young and 17 aged MII eggs, imaged as in (B).(D) Age-related increase in centromere distortion (circularity < 0.80) in MII eggs from young and old mice, imaged as in (B).(E) Representative chromosome spread of a young MII mouse egg. DNA signal (blue, Picogreen, confocal mode), outer kinetochore region (white, Hec1, confocal mode), and the centromeric signal (green, CENP-A, STED mode) are shown. Right: one centromere in each panel (STED microscopy, grayscale; A and B, sister centromeres).(F) Representative chromosome spread of an MII egg from an old mouse (63 weeks), labeled as in (E).(G) Surface area of CENP-A centromeric domains in MII eggs from young and aged mice, acquired as in (E) and (F). Distortion of the centromeric domain was measured as the surface area of the smallest circle encompassing the CENP-A signal (119 young and 221 aged centromeres analyzed). Mean and SD are shown.(H) Representative Airyscan-imaged metaphase-II chromosome from an aged mouse egg (61-week-old female). Outer kinetochores (white, Hec1), centromeres (green, CENP-A), and DNA (blue, Hoechst) are shown. Scale bars represent 1 μm in overview, and 0.5 μm in insets.(I) Quantification of kinetochore configurations visualized with Airyscan microscopy, based on the outer Hec1 signal in 37 MII mouse eggs (3 experiments), labeled as in (H).(J) Quantification of how frequently the MII outer kinetochore pattern follows changes in the underlying centromeric domain. Concordant indicates centromere/outer kinetochore pairs where the CENP-A/Hec1 signals were both either compact or fragmented. 740 centromeres and their corresponding kinetochores from 19 old MII mouse eggs (3 experiments), labeled as in (B) and (H).Young MII eggs: 8-week-old females; aged eggs (12 animals): 60- to 64-week-old females. Scale bars represent 4 μm in overviews, and 0.5 μm in insets in (E) and (F).p values are designated as ∗p < 0.05 and ∗∗∗∗p < 0.0001. p values were calculated with Student’s t test (I) and Fisher’s exact test (D). In (A), mean fluorescence measurements per egg were compared by one-way ANOVA followed by Tukey’s test. Error bars show SEM. White arrows point to compact kinetochores, and yellow arrows point to lobes within a fragmented MII kinetochore.See also Figure S1.

Fig 4: Fragmented Kinetochores Are More Likely to Be Abnormally Attached to Spindle Microtubules(A) Quantification of the number of distinct k fibers attaching to compact/fragmented kinetochores at metaphase II (28 aged mouse MII eggs, 3 experiments) is shown.(B) Representative kinetochore-microtubule attachment types, relative to the kinetochore fragmentation status. The scale bar represents 1 μm.(C) Quantification of the microtubule attachment types shown in (B) across the specified MII kinetochore categories on intact chromosomes (28 aged mouse MII eggs, 3 experiments).(D) Representative merotelically attached single chromatid and its respective kinetochore, aligned at the equator of a metaphase-II spindle in an aged mouse egg. Scale bars represent 5 μm in overview, and 1 μm in insets.(E) Quantification of the subtypes of merotelic attachments on fragmented kinetochores, relative to the chromosome architecture and the site of microtubule attachment.(F) Occurrence of kinetochore architectures following Trim-Away of Smc3 in mouse MII eggs subjected to either DMSO or low-dose nocodazole (62 young MII eggs, 3 experiments).(G) Representative chromosome spreads of control and Smc3 partially depleted MI oocytes from young mice subjected to monastrol/control DMSO drug treatments. Each inset shows two sister kinetochores/centromeres only. Outer kinetochores (white, Hec1), centromeres (green, CENP-A), and chromosomes (blue, Hoechst) are shown. Scale bars represent 5 μm in overview, and 1 μm in insets.(H) Occurrence of kinetochore architectures in MI oocytes from young mice treated as in (G). Quantifications are based on Hec1 labeling in 80 MI oocytes (2 experiments).DNA (blue, Hoechst), microtubules (green, α-tubulin), and kinetochores (magenta, CREST) are shown in (B) and (D).p values are designated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. p values in (F) were calculated with Student’s t test and in (A), (C), and (E) with Fisher’s exact test. In (H), mean measurements per oocyte were compared by one-way ANOVA followed by Tukey’s test. Error bars show SEM. Arrows: white point to compact kinetochores, and yellow/blue to lobes within fragmented kinetochores/centromeres, respectively.See also Figure S7.

Fig 5: Cohesin Loss Causes Decompaction of Centromeric Chromatin and Fragmentation of Meiotic Kinetochores(A) Scheme of Trim-Away experiments to acutely degrade cohesins in mouse metaphase-II eggs. Trim-Away is a protein depletion tool, which uses antibodies to rapidly remove unmodified, native proteins via the antibody receptor/E3 ubiquitin ligase TRIM21 and the endogenous protein degradation machinery.(B) Immunolabeled young TRIM21 overexpressing metaphase-II mouse eggs, microinjected with either a control IgG antibody (top) or an anti-Rec8 antibody provided in excess (bottom), as in (A). Images are z projections of 63–69 sections, acquired every 0.19 μm. Microtubules (green, α-tubulin) and chromosomes (blue, Hoechst). The scale bar represents 5 μm.(C) Representative chromosome spreads from young metaphase-II mouse eggs depleted of Smc3. Insets: a chromosome (control, left panel) or individual single chromatids (all other panels). Outer kinetochores (white, Hec1), centromeres (green, CENP-A), and chromosomes (blue, Hoechst) are shown.(D) Normalized diameter of the CENP-A centromeric domain in control and young mouse MII eggs depleted for Smc3, as in (C). CENP-A domains were divided into three groups, based on the fragmentation status of the overlying Hec1 kinetochore. 723 centromeres from 20 MII eggs were evaluated (2 experiments).(E) Occurrence of different kinetochore architectures based on outer kinetochore Hec1 signal in cells treated as in (C). Data are from 35 MII eggs (young mice; 2 experiments).(F) Normalized diameter of the Hec1 kinetochore domain in control and young mouse eggs depleted for Smc3. Both the fragmentation status and the diameter measurements were based on Hec1 labeling. 755 kinetochores from 20 MII eggs were evaluated (2 experiments).(G) The diameter of the CENP-A centromeric domain (a.u.) plotted against the Hec1 outer kinetochore diameter (a.u.) for each of the 565 MII kinetochores following a full depletion of Smc3/control MII eggs as in (C). Pearson’s correlation coefficients: r = 0.402 (controls) and r = 0.495 (experimental group).(H) Inner plates of MII kinetochores in metaphase-arrested eggs from young mice in a control chromosome (left panel; two sister kinetochores) and in single chromatids induced by a full depletion of Rec8 (other panels), achieved as in (B). Chromosomes (blue, Hoechst) and kinetochore inner plates (magenta, CREST) are shown.(I) Occurrence of MII kinetochore configurations in young eggs following the targeting of distinct cohesin subunits (Rec8, Smc3) by Trim-Away (full depletion) and in control eggs. Data are from 79 MII eggs from young mice (3 experiments).(J) Young metaphase-II eggs labeled with the inner kinetochore protein CENP-C. Images show a control chromosome (left panel; two sister kinetochores) and single chromatids induced by a full depletion of Rec8 (other panels), obtained as in (A). Chromosomes (blue, Hoechst) and kinetochore inner plates (yellow, CENP-C) are shown.(K) Occurrence of different kinetochore architectures based on the inner kinetochore CENP-C signal in cells treated as in (J). Data from 36 MII eggs (young mice; 2 experiments).Scale bars represent 2 μm in overviews, and 0.5 μm in insets in (C), (H), and (J).p values are designated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. p values were calculated with Student’s t test in (I) and Fisher’s exact test in (E) and (K); in (D) and (F), mean measurements per egg were compared by one-way ANOVA followed by Tukey’s test. Error bars show SEM. Box plots show median (horizontal lines), mean (small squares), 25th and 75th percentiles (boxes), and 5th and 95th percentiles (whiskers). Arrows: white point to compact kinetochores, and yellow point to lobes within a fragmented MII kinetochore.See also Figures S3–S5 and Videos S1 and S2.