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.