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, 28 (9), 1344-1356.e5

Chromosome Segregation Is Biased by Kinetochore Size

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Chromosome Segregation Is Biased by Kinetochore Size

Danica Drpic et al. Curr Biol.

Abstract

Chromosome missegregation during mitosis or meiosis is a hallmark of cancer and the main cause of prenatal death in humans. The gain or loss of specific chromosomes is thought to be random, with cell viability being essentially determined by selection. Several established pathways including centrosome amplification, sister-chromatid cohesion defects, or a compromised spindle assembly checkpoint can lead to chromosome missegregation. However, how specific intrinsic features of the kinetochore-the critical chromosomal interface with spindle microtubules-impact chromosome segregation remains poorly understood. Here we used the unique cytological attributes of female Indian muntjac, the mammal with the lowest known chromosome number (2n = 6), to characterize and track individual chromosomes with distinct kinetochore size throughout mitosis. We show that centromere and kinetochore functional layers scale proportionally with centromere size. Measurement of intra-kinetochore distances, serial-section electron microscopy, and RNAi against key kinetochore proteins confirmed a standard structural and functional organization of the Indian muntjac kinetochores and revealed that microtubule binding capacity scales with kinetochore size. Surprisingly, we found that chromosome segregation in this species is not random. Chromosomes with larger kinetochores bi-oriented more efficiently and showed a 2-fold bias to congress to the equator in a motor-independent manner. Despite robust correction mechanisms during unperturbed mitosis, chromosomes with larger kinetochores were also strongly biased to establish erroneous merotelic attachments and missegregate during anaphase. This bias was impervious to the experimental attenuation of polar ejection forces on chromosome arms by RNAi against the chromokinesin Kif4a. Thus, kinetochore size is an important determinant of chromosome segregation fidelity.

Keywords: Indian muntjac; aneuploidy; centromere; chromosome congression; chromosome segregation; kinetochore; merotelic attachments; mitosis.

Figures

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Figure 1
Figure 1
Centromere and Kinetochore Functional Layers Scale Proportionally with Centromere Size (A) Normal karyotype of female Indian muntjac fibroblasts. Scale bar, 5 μm. (A′) Centromere length of Indian muntjac chromosomes (box-whisker plots, n = 40 cells, ∼80 kinetochores per chromosome type, Mann-Whitney rank-sum test, p < 0.001 for all comparisons). (B–G) Immunofluorescence of Indian muntjac chromosome spreads (blue) and the centromere and kinetochore proteins (green) CENP-A (B), Ndc80 (C), CENP-E (D), Mad2 (E), pAurora B (F), and pKNL1 (G). Scale bars, 5 μm. (B′–G′) Respective quantification of protein levels at Indian muntjac kinetochores, relative to chromosome 3+X (C3X) for CENP-A (B′), Ndc80 (C′), CENP-E (D′), Mad2 (E′), pAurora B (F′), and pKNL1 (G′) (mean ± SD, n ≥ 37 cells per condition, ∼100 kinetochores per chromosome type, ∗∗∗p < 0.001 relative to controls, Mann-Whitney rank-sum test).
Figure 2
Figure 2
Indian Muntjac Kinetochores Show Typical Structural Organization and Their Microtubule Binding Capacity Scales with Kinetochore Size (A) Selected optical planes from an Indian muntjac fibroblast stably expressing Centrin-1-GFP to label the centrioles (green), showing kinetochore pairs for C3X (A′) and neighbor chromosome with smaller (A″) centromere. The inner and outer parts of the kinetochores were delineated by CENP-A (green) and Ndc80/Hec1 (red). DNA was counterstained with DAPI (blue). (A′ and A″) Higher-magnification views of C3X (A′) and smaller kinetochores (A″). Dashed lines denote where intra-kinetochore distances were measured. Scale bars, 5 μm (A) and 1 μm (A′ and A″). Differences between large and small kinetochores were not statistically significant (t test). KT, kinetochore. (B) Single electron microscopy section from consecutive series highlighting the standard organization of the Indian muntjac centromere and kinetochore plates. L1 and L2 correspond to the plates on chromosome C3X; S1 and S2 correspond to the plates on a neighboring chromosome with smaller kinetochores. Scale bar, 2 μm. (C) Z projection of the entire volume of the corresponding series shown in Figure S1. K fibers on the C3X chromosome comprise a larger number of microtubules (green). Kinetochore plates (magenta) and chromosomes (blue) are indicated. Scale bar, 1 μm. MT, microtubule. (D) Surface-rendered model of the volume shown in Figure S1. C3X kinetochores are approximately twice as large as in chromosomes with smaller kinetochores. (E) Quantification of the number of attached microtubules as a function of the approximate kinetochore area. Plot shows serial-section electron microscopy data from 26 kinetochores from 13 chromosomes and 3 cells. See also Figure S1.
Figure 3
Figure 3
The Molecular Landscape Required to Establish Functional Kinetochore-Microtubule Attachments Is Conserved in Indian Muntjac (A) Live-cell imaging of Indian muntjac fibroblasts stably expressing H2B-GFP to visualize the chromosomes (green) and treated with 50 nM SiR-tubulin to label spindle microtubules (magenta). Ndc80, Mad2, Mps1, Clasp1, and Survivin were knocked down by RNAi. Scale bars, 5 μm. Time, hr:min. (B) Mitotic timing of Indian muntjac fibroblasts stably expressing H2B-GFP with or without addition of 50 nM SiR-tubulin. There is no statistically significant difference in mitotic timing from NEB to anaphase onset (ANA) in the presence or absence of SiR-tubulin (Mann-Whitney rank-sum test, p = 0.591). n.s., not significant. (C) Protein lysates obtained after RNAi were immunoblotted with an antibody specific to each protein of interest. GAPDH was used as loading control. See also Video S1.
Figure 4
Figure 4
Any Chromosome May Use Either the CENP-E-Dependent or CENP-E-Independent Pathway to Congress, Regardless of Kinetochore Size (A) Control Indian muntjac fibroblasts stably expressing CENP-A-GFP (green) and treated with 20 nM SiR-tubulin (magenta). Scale bar, 5 μm. Time, hr:min. (B) Indian muntjac fibroblasts stably expressing CENP-A-GFP (green) and treated with 20 nM SiR-tubulin (magenta) after CENP-E inhibition with 20 nM GSK923295. Scale bars, 5 μm. Time, hr:min. n = 28 cells, pool of six independent experiments. The arrows indicate the position of large kinetochores from C3X chromosomes. See also Figure S2 and Video S2.
Figure 5
Figure 5
Chromosome Congression and Bi-orientation in Indian Muntjac Are Biased by Kinetochore Size (A) Immunofluorescence of an Indian muntjac fibroblast after CENP-E inhibition showing chromosomes (DAPI; white in merged image), kinetochores (ACA, white; green in merged image), and microtubules (α-tubulin; magenta in merged image). Scale bar, 5 μm. (B) Quantification of the number of chromosomes with small or large kinetochores at the pole after CENP-E inhibition by immunofluorescence in fixed cells (magenta and green lines) and respective theoretical prediction based on a binomial distribution (gray bars). (C) Probability of each individual chromosome with small or large kinetochores to stay at the pole upon CENP-E inhibition (arbitrary units) (mean ± SD, n = 621 cells, six independent experiments, p = 0.0067, t test). (D) 4D (x, y, z, t) tracking of chromosomes with large kinetochores after CENP-E inhibition to determine their position relative to the poles at NEB and the forming mitotic spindle (see dashed box in A for reference). Note that chromosomes with large kinetochores are randomly distributed relative to the equator and the spindle poles. See also Figure S2 and Video S3.
Figure 6
Figure 6
Chromosomes with Larger Kinetochores Are More Prone to Establish Erroneous Merotelic Attachments that Result in Non-random Missegregation (A) Error correction after monastrol washout in live Indian muntjac fibroblasts stably expressing CENP-A-GFP (green) and treated with 20 nM SiR-tubulin (magenta). Dashed boxes highlight a region with a chromosome with large kinetochores (arrows in lower panels that show 1.5× zoom images, plus additional time frames). Scale bar, 5 μm. Time, min:s. (B) STED/confocal image of a prometaphase Indian muntjac fibroblast after monastrol washout showing syntelic attachments. Microtubules (α-tubulin, magenta), chromosomes (DAPI, white), and kinetochores (ACA, green) are indicated. Scale bar, 5 μm. (C) Quantification of erroneous attachments on chromosomes with small or large kinetochores (KTs) (n = 207 cells, pool of three independent experiments). (D) Frequency of anaphase cells with lagging chromosomes in controls and after monastrol washout (mean ± SD; each data point indicates an independent experiment; 2,099 control anaphase cells scored; 3,739 anaphase cells scored after monastrol washout; Mann-Whitney rank-sum test). (E) STED/confocal image of an Indian muntjac fibroblast in anaphase after monastrol washout. Microtubules (α-tubulin, magenta), chromosomes (DAPI, white), and kinetochores (ACA, green) are indicated. Dashed boxes indicate a lagging chromatid (C3X(a)) containing a large kinetochore with merotelic attachments and the corresponding sister (C3X(b)). Scale bar, 5 μm. The images and graphical sketches on the right highlight the type of attachments in the two sisters (2× zoom). (F) Frequency of anaphase cells with at least 1 lagging chromosome with small or large kinetochores after monastrol washout (n = 32 cells from nine independent experiments). Dashed bar represents theoretical values for the frequency of lagging chromosomes with small kinetochores, if the probability to lag was equal for chromosomes with large or small kinetochores. (G) Live-cell imaging of an Indian muntjac fibroblast stably expressing H2B-GFP (green) and treated with 50 nM SiR-tubulin (magenta) illustrating missegregation of lagging chromosomes after monastrol washout. Scale bars, 5 μm. Time, hr:min. The green and magenta arrows indicate two lagging chromosomes that failed to integrate and reintegrated the main nucleus, respectively. (H) Percentage of cells (from live-cell imaging) with lagging chromosomes incorporating or forming micronuclei after monastrol washout (n = 59 cells, pool of five independent experiments). See also Figures S3–S5 and Videos S4, S5, and S6.
Figure 7
Figure 7
Polar Ejection Forces on Chromosome Arms Ensure Mitotic Fidelity but Are Not Implicated in the Observed Missegregation Bias for Chromosomes with Large Kinetochores (A) Live-cell imaging of Indian muntjac fibroblasts stably expressing H2B-GFP to visualize the chromosomes (green) in control (top) and Kif4a RNAi (bottom) cells treated with 50 nM SiR-tubulin to label spindle microtubules (magenta). Scale bar, 5 μm. Time, hr:min. White arrows point to the chromosome arms facing the spindle poles. (B) Western blot to monitor Kif4a levels after RNAi. GAPDH was used as loading control. (C) Chromosome missegregation after Kif4a RNAi (fixed cells). Kinetochores (anti-ACA), α-tubulin, and DNA (DAPI) are indicated. Scale bars, 5 μm. (D and E) Comparison of the frequency of anaphase cells with lagging chromosomes in live (D) and fixed (E) material after Kif4a depletion and/or monastrol washout. (F) Frequency of anaphase cells with at least 1 lagging chromosome with small or large kinetochores after monastrol washout in Kif4a-depleted fibroblasts. Dashed bar represents theoretical values for frequencies of lagging chromosomes with small kinetochores if the probability to lag was equal for chromosomes with small or large kinetochores. See also Video S7.

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