Edward D. Salmon

The spindle assembly checkpoint delays anaphase until all chromosomes are attached to a mitotic spindle. The mad (mitotic arrest-deficient) and bub (budding uninhibited by benzimidazole) mutants of budding yeast lack this checkpoint and fail to arrest the cell cycle when microtubules are depolymerized. A frog homolog of MAD2 (XMAD2) was isolated and found to play an essential role in the spindle assembly checkpoint in frog egg extracts. XMAD2 protein associated with unattached kinetochores in prometaphase and in nocodazole-treated cells and disappeared from kinetochores at metaphase in untreated cells, suggesting that XMAD2 plays a role in the activation of the checkpoint by unattached kinetochores. This study furthers understanding of the mechanism of cell cycle checkpoints in metazoa and provides a marker for studying the role of the spindle assembly checkpoint in the genetic instability of tumors.

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 microM 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.

In this article, we review the dynamic nature of the filaments (microtubules) that make up the labile fibers of the mitotic spindle and asters, we discuss the roles that assembly and disassembly of microtubules play in mitosis, and we consider how such assembling and disassembling polymer filaments can generate forces that are utilized by the living cell in mitosis and related movements.