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| milestone 18
Mad and Bub check it out
During the cell cycle, a cell goes through a series of sequential events — one step has to be completed before the next is initiated. This linear progression can be achieved in at least two ways: each step could require a product of the preceding step for its initiation, or there might be regulatory feedback mechanisms which ensure that a subsequent step in the cell cycle is not initiated if a crucial event, known as a checkpoint, has not been completed successfully. In 1991, two studies published in Cell, one led by Andrew Hoyt, the other by Andrew Murray, identified the spindle checkpoint by showing the existence of a feedback-control mechanism that prevents cells from leaving mitosis if their mitotic spindle has been incompletely assembled. Using independent approaches, the two groups isolated mutants that exit mitosis and proceed to the next phase of the cell cycle, despite defects in their spindle formation.
The existence of a checkpoint mechanism that involves Rad9 (see Milestone 14) and controls the state of genomic DNA had already been shown. Could a similar mechanism operate during mitosis? Components of a feedback mechanism can be isolated if conditions are found under which cells overcome cell-cycle arrest. The two groups carried out genetic screens designed to address the possibility that exit from mitosis depends on microtubule function. Mutagenized Saccharomyces cerevisiae cells that were grown in the presence of benomyl, an inhibitor of microtubule polymerization, were screened for mutants that failed to arrest in mitosis. The five mutants isolated by Li et al. mapped to three loci and were named 'mitotic arrest-deficient', or mad. By careful analysis of nuclear morphology and the DNA content, the authors showed that mad cells die at the end of nuclear division, presumably when they attempt to segregate their chromosomes in the absence of a functional spindle. Benomyl slows down microtubule assembly, and the mad mutant phenotype can be rescued if DNA synthesis is slowed down experimentally. It increases the time between the onset of spindle formation and the end of mitosis — so, for mad mutants grown on benomyl, it simply means more time for spindle assembly. By 1991, it was known that entry to mitosis is regulated by a protein kinase — the so-called 'maturation-promoting factor' (MPF; see Milestone 7). Activation of MPF induces entry into mitosis and assembly of the mitotic spindle; conversely, its inactivation induces interphase, chromosome segregation and cell division. By looking at activated MPF in mad cells as they proceed through the cell cycle, Li et al. showed that, whereas in normal cells grown on benomyl cell-cycle arrest coincides with high levels of MPF, mad cells fail to stabilize their MPF levels under the same conditions. It is the drop in MPF activity that pushes mad cells towards exit from mitosis, despite their spindle defect. As might be expected, some mad cells show extensive chromosome loss, even without benomyl, which indicates that their wild-type products are needed for accurate chromosome segregation. Cloning of Mad2 showed a largely novel protein that contained calcium-binding motifs. Hoyt and colleagues chose to study three mutants that were recovered from their screens; named bub, for 'budding uninhibited by benzimidazole' — a benomyl-related compound. Despite unsuccessful completion of mitosis, bub mutants proceed to the next cell cycle and initiate DNA replication, duplicate their spindle pole and bud. Hoyt et al. confirmed the microtubule-specific phenotype of bub mutants by crossing them to an α-tubulin mutant, thereby replacing the chemical microtubule-disruption method with a genetic one. But the authors also showed that disrupting microtubule structure alone was not enough to kill bub mutants — progression through the cell cycle was absolutely required for their death. Similar to mad mutants, bub mutants also affect the cell cycle by regulating MPF activity. Identification of mad and bub mutants indicated, for the first time, the existence of a mitotic-spindle checkpoint that was linked to the regulation of MPF activity — crucial for the decision of whether to arrest or progress through the cell cycle. At the time it was not clear how Mad and Bub regulated MPF levels, and another player involved in the spindle checkpoint — Mps1 — was yet to be discovered. The mad and bub mutants also provided the essential components of the mechanism by which cells ensured equal chromosome segregation to the two daughter cells — a process fundamental to genomic stability. This work formed the basis of the current model for how the mitotic checkpoint regulates the activity of the anaphase-promoting complex (see figure and Milestone 20).
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