The existence of critical 'triggers' or 'points of no return' at key cell-cycle transitions was postulated by Mazia as early as 1961 [2
], although, as he later acknowledged in 1987, the concept as originally formulated proved non-productive. The problem was that the triggers were envisaged to be essential internal components of the molecular cascades that drive the cell cycle. Checkpoints, by contrast, are external control mechanisms that are not required for forward progression [3
]. Thus, a fundamental feature of a checkpoint is that its activities are not manifested under conditions in which the potential for errors is minimal: only when conditions become stressful and errors are likely to occur do checkpoints become essential survival tools. This criterion formed the basis of early screens to identify mitotic checkpoint components in yeast [4
]. The name of three well known mitotic checkpoint proteins, Mad1-3, comes from the acronym 'Mitotic Arrest Deficient', reflecting the fact that Mad
mutants progress through mitosis with similar kinetics whether or not the spindle is present (and thus in the presence of unattached kinetochores, which normally arrest mitosis - see legend to Figure ). In contrast, wild-type cells arrest in mitosis when spindle formation is inhibited with microtubule poisons. Under normal conditions, however, both wild-type and Mad-deficient cells or organisms with low chromosome number and efficient spindle assembly mechanisms (for example, yeast and Drosophila
) grow equally well, which reflects the fact that the mitotic checkpoint is not essential when the frequency of errors is naturally low.
Some argue that the function of the mitotic checkpoint in yeast and Drosophila
is different from that in mammals, because in mammals inactivation of checkpoint genes is lethal even in the absence of other stresses. This argument is conceptually flawed, as the fate difference observed simply reflects differences in the speed of spindle assembly. Because of the stochastic nature of interaction between kineto chores and spindle microtubules, the presence of numerous chromosomes and/or centrosomes (spindle poles) greatly increases the time required for spindle assembly. Under this condition, unless mitotic exit is delayed by the checkpoint until all kinetochores have attached to the spindle the progeny will be aneuploid. For this reason, inactivating the mitotic checkpoint in mammals results in a rapid rise in aneuploidy and ultimately death [5
]. A nice illustration of the interplay between the checkpoint, the kinetics of spindle assembly, and cell/organism viability comes from recent work in Drosophila
that accumulate supernumerary centrosomes. Although this condition itself does not compromise viability, it slows the rate of spindle assembly. Predictably, eliminating the mitotic checkpoint by deleting Mad2, which has no effect on wild-type Drosophila
, becomes lethal in flies with supernumerary centrosomes [6
]. It is important to emphasize that in the latter case, cells are not 'killed by the checkpoint' as sometimes described in the literature. Instead, cells die because they ultimately become highly aneuploid in the absence of a functional checkpoint. The function of the mitotic checkpoint is to prevent premature mitotic exit - and nothing else.
The failure to distinguish true checkpoint proteins from those involved in the pathway targeted by the mitotic checkpoint is common, and usually results from too narrow a focus on molecular interactions without regard for the conceptual context. It is obvious that checkpoint proteins must interact not only with the structure or event being monitored (for example, an unattached kinetochore), but also with the pathway and structures whose activity is required to drive cell-cycle progression. This being the case, because the checkpoint itself is not required for forward progression, proteins whose mutations prolong mitosis can never be considered true checkpoint components.
For example, exit from mitosis requires the activation of a large ubiquitin ligase, termed the anaphase-promoting complex (APC) or cyclosome, which tags for destruction proteins that hold replicated chromosomes together or that keep the cell in mitosis (these include securin and cyclin B; Figure ). The APC is activated by an activator protein, Cdc20, and if Cdc20 is depleted, the cell arrests in mitosis. Despite the fact that Cdc20 interacts directly with bona fide checkpoint proteins (for example, Mad2), this phenotype clearly demonstrates that Cdc20 itself is not a checkpoint protein. Interaction between Cdc20 and checkpoint proteins is expected as the goal of the mitotic checkpoint is, biochemically, to prevent premature activation of the APC by sequestering Cdc20. Mistakenly considering Cdc20 or other proteins intrinsically required for forward mitotic progression to be directly involved in the checkpoint degrades the checkpoint concept back to Mazia's internal 'triggers'.