The spindle checkpoint
Introduction
During mitosis, the two daughter cells receive one copy of each chromosome. Sister chromatids attach via a specialized DNA–protein complex, known as the kinetochore, to microtubules emanating from opposite poles of the mitotic spindle. When and only when all kinetochores have bound microtubules, the mitotic spindle proceeds to pull the sister chromatids apart. Cells go to great lengths to ensure that each daughter cell receives one and only one copy of each chromosome. A surveillance mechanism, known as the spindle checkpoint, is essential for ensuring fidelity in chromosome transmission. The checkpoint monitors correct attachment of kinetochores to microtubules and inhibits sister chromatid separation and thus onset of anaphase when a defect is detected 1, 2.
Genetic screens in budding yeast have identified several components of the spindle checkpoint (Table 1). Subsequently, homologs have been identified in higher eukaryotes showing that the spindle-checkpoint pathway is highly conserved (Table 1). Yeast cells carrying a mutation in any of the genes encoding a component of the spindle checkpoint attempt sister chromatid segregation despite mitotic spindle defects or improper attachment of kinetochores to microtubules, the result of which is disastrous, with cells either losing or acquiring extra chromosomes. Loss of chromosomes in haploid cells leads to cell death. In diploid cells, it can either cause cell death or the accumulation of cancer-causing mutations.
The spindle checkpoint halts cell-cycle progression prior to sister-chromatid separation. Sister-chromatid separation is triggered by ubiquitin-mediated degradation of regulators of sister-chromatid cohesion. A multisubunit ubiquitin ligase known as the cyclosome or anaphase promoting complex (APC [3]) ubiquitinates regulators of sister-chromatid segregation: Pds1 in budding yeast and Cut2 in fission yeast, leading to their destruction by the proteasome. APC-dependent proteolysis is not only important for sister-chromatid separation at the metaphase–anaphase transition but also for a second step in mitosis. APC-dependent proteolysis participates in triggering exit from mitosis by degrading mitotic cyclins, the regulatory subunits of the Cdc2-mitotic kinases [3].
As sister-chromatid separation and exit from mitosis are initiated by APC-dependent proteolysis, it is activation of this degradation machinery or access to its substrates that must be inhibited in order to prevent cell-cycle progression. In the past year, work in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Xenopus and mammalian cell lines has revealed how the spindle-checkpoint pathway prevents progression into anaphase in response to spindle damage. The checkpoint prevents the APC activator Cdc20/Slp1 from activating the ubiquitin ligase activity of the APC. This major breakthrough in our understanding of how the spindle checkpoint impinges on the cell cycle will be the focus of my review.
Section snippets
The signal
The spindle checkpoint can sense a multitude of spindle defects, ranging from the presence of a single unattached kinetochore to massive defects induced by microtubule-depolymerising drugs such as nocodazole. It is now thought that all these defects cause perturbation of microtubule–kinetochore attachment and that it is these defects in attachment that are sensed by the checkpoint.
There are two models for the generation of the signal by unattached kinetochores that leads to activation of the
The signaling cascade
Protein phosphorylation probably plays an important role in transmitting the signal generated by an unattached kinetochore. Genetic studies in budding yeast have identified two protein kinases, MPS1 and BUB1, as being required for cell-cycle arrest in response to spindle defects, and a protein, Mad1, that is specifically phosphorylated in response to activation of the spindle checkpoint 13, 14, 15. The finding that Mad1 is phosphorylated in a checkpoint-dependent manner [15] and that
The checkpoint target
The WD40 repeat protein Cdc20 (called Slp1 in fission yeast) was shown to be an activator of APC-dependent proteolysis by genetic means in budding yeast 23, 24. Cdc20 primarily targets APC substrates such as Pds1, the inhibitor of sister-chromatid separation, for degradation at the metaphase–anaphase transition, whereas Cdh1/Hct1, a relative of Cdc20 is more important for degrading mitotic cyclins and other APC substrates during exit from mitosis and G1 23, 24, 25. Biochemical analysis in
Conclusions
Work over the past year in diverse systems has greatly enhanced our understanding about the spindle checkpoint and how spindle damage prevents cell-cycle progression. Components of the spindle checkpoint were found to inhibit the activity of Cdc20–APC complexes by directly binding to them. The finding that these complexes also exist in cells that are not treated with microtubule poisons raises the possibility that the checkpoint acts during each cell cycle.
Challenges for the future include
Acknowledgements
I am grateful to Steve Elledge and Peter Sorger for helpful comments and critical reading of the manuscript.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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