Merotelic Kinetochore Orientation Is a Major Mechanism of Aneuploidy in Mitotic Mammalian Tissue Cells

TLDR

During mitosis, lagging chromosomes that remain near the spindle equator after anaphase onset represent a segregation error. Lagging chromosomes are single chromatids whose kinetochores attach merotelically to microtubules from opposite poles, a configuration confirmed by electron microscopy and exacerbated by nocodazole‑induced kinetochore expansion. In PtK1 cells, lagging chromosomes occur in 1.16 % of untreated anaphases and rise to 17.55 % after nocodazole arrest; their stretched, merotelic kinetochores lack checkpoint signals, demonstrating that merotelic orientation is a major, checkpoint‑undetected source of aneuploidy.

Abstract

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 μM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2–5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.