Chromosome missegregation in human cells arises through specific types of kinetochore-microtubule attachment errors

Abstract

Most solid tumors are aneuploid, and many missegregate chromosomes at high rates in a phenomenon called chromosomal instability (CIN). CIN reflects the erosion of mitotic fidelity, and it correlates with poor patient prognosis and drug resistance. The most common mechanism causing CIN is the persistence of improper kinetochore-microtubule attachments called merotely. Chromosomes with merotelic kinetochores often manifest as lagging chromosomes in anaphase, suggesting that lagging chromosomes fail to segregate properly. However, it remains unknown whether the lagging chromosomes observed in anaphase segregate to the correct or incorrect daughter cell. To address this question, we tracked the segregation of a single human chromosome during cell division by using LacI-GFP to target an integrated LacO array. By scoring the distribution of each sister chromatid during mitosis, we show that a majority of lagging chromosomes in anaphase segregate to the correct daughter cell. Instead, sister chromatids that segregate erroneously frequently do so without obvious evidence of lagging during anaphase. This outcome is expected if sister kinetochores on a chromosome bind microtubules oriented toward the same spindle pole, and we find evidence for syntelic kinetochore attachments in cells after treatments that increase missegregation rates. Thus, lagging chromosomes in anaphase are symptomatic of defects in kinetochore-microtubule attachment dynamics that cause chromosome missegregation associated with CIN, but the laggards rarely missegregate.

Fig. 1.

Fates of sister chromatids of a single chromosome during mitosis. Corresponding daughter HCT116 cells expressing lacIGFP with LacO arrays integrated into a single chromosome were fixed and imaged after inducing high levels of merotely with MCAK siRNA or monastrol washout. Schematic of possible segregation fates of the marked chromosome (Upper) and representative images of each fate (Lower) are shown. (Scale bar: 10 μm.)

Fig. 2.

Fates of sister chromatids of a single chromosome during anaphase. HCT116 cells in anaphase expressing lacIGFP with LacO arrays integrated into a single chromosome were fixed and imaged after inducing high levels of merotely with MCAK siRNA or monastrol washout. Representative images show the marked chromosome segregating correctly, with one chromosome in each anaphase plate (A), lagging in the spindle midzone (B), or missegregating with no evidence of lagging (C). Arrowheads point to the LacIGFP labeled chromosome. (Scale bar: 5 μm.)

Fig. 3.

Various kinetochore–microtubule attachments. RPE1 cells recovering from monastrol treatment to induce k-MT attachment errors were extracted in the presence of calcium; fixed; stained for kinetochores (Hec1) in red, microtubules (green), and DNA (blue); and imaged by using confocal microscopy to identify k-MT attachments. Single focal planes showing amphitely (A), merotely (B), and syntely (C) are shown in Insets. (Scale bars: Left, 2.5 μm; Right, 1 μm.)

Fig. 4.

Kinetochore microtubule attachments and chromosome fate. Schematic shows numerical microtubule differences in merotelic attachments (pauci, few; equi, equal; multi, many) and the likely fate of these attachment errors if they persist into anaphase.