The spindle checkpoint

Prior to sister-chromatid separation, the spindle checkpoint inhibits cell-cycle progression in response to a signal generated by mitotic spindle damage or by chromosomes that have not attached to microtubules. Recent work has shown that the spindle checkpoint inhibits cell-cycle progression by direct binding of components of the spindle checkpoint pathway to components of a specialized ubiquitin-conjugating system that is responsible for triggering sister-chromatid separation.     

The spindle assembly checkpoint

The spindle assembly checkpoint monitors proper chromosome attachment to spindle microtubules and is conserved from yeast to humans. Checkpoint components reside on kinetochores of chromosomes and show changes in phosphorylation and localization as cells proceed through mitosis. Adaptation to prolonged checkpoint arrest can occur by inhibitory phosphorylation of Cdc2.     

Visualizing the spindle checkpoint in Drosophila spermatocytes

The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila.     

Flies without a spindle checkpoint

Mad2 has a key role in the spindle-assembly checkpoint (SAC) - the mechanism delaying anaphase onset until all chromosomes correctly attach to the spindle. Here, we show that unlike every other reported case of SAC inactivation in metazoans, mad2-null Drosophila are viable and fertile, and their cells almost always divide correctly despite having no SAC and an accelerated 'clock', which is caused by premature degradation of cyclin B. Mitosis in Drosophila does not need the SAC because correct chromosome attachment is achieved very rapidly, before even the cell lacking Mad2 can initiate anaphase. Experimentally reducing spindle-assembly efficiency renders the cells Mad2-dependent. In fact, the robustness of the SAC may generally mask minor mitotic defects of mutations affecting spindle function. The reported lethality of other Drosophila SAC mutations may be explained by their multifunctionality, and thus the 'checkpoint' phenotypes previously ascribed to these mutations should be considered the consequence of eliminating both the checkpoint and a second mitotic function.     

The spindle checkpoint in the dinoflagellate Crypthecodinium cohnii

Dinoflagellates are a major group of organisms with an extranuclear spindle. As the purpose of the spindle checkpoint is to ensure proper alignment of the chromosomes on the spindle, dinoflagellate cell cycle control may be compromised to accomodate the extranuclear spindle. In the present study, we demonstrated that nocodazole reversibly prolonged the G2 + M phase of the dinoflagellate cell cycle, in both metaphase and anaphase. The regulation of the spindle checkpoint involves the activation and inhibition of the anaphase promoting complex (APC), which in turn degrades specific cell cycle regulators in the metaphase to anaphase transition. In Crypthecodinium cohnii, nocodazole was also able to induce a prolongation of the degradation of mitotic cyclins and a delay in the inactivation of p13(suc1)-associated histone kinase activities. In addition, cell extracts prepared from C. cohnii in G1 phase and G2/M phase (or nocodazole treated) were able to activate and inhibit, respectively, the degradation of exogenous human cyclin B1 in vitro. The present study thus demonstrated the presence of the spindle checkpoint and APC-mediated cyclin degradation in dinoflagellates. This is discussed in relation to a possible role of the nuclear membrane in mitosis in dinoflagellates.     

Linking kinetochore-microtubule binding to the spindle checkpoint

The spindle checkpoint blocks cell-cycle progression until chromosomes are properly attached to the mitotic spindle. Popular models propose that checkpoint proteins associate with kinetochores to produce a "wait anaphase" signal that inhibits anaphase. Recent data suggest that a two-state switch results from using the same kinetochore proteins to bind microtubules and checkpoint proteins. At least eight protein kinases are implicated in spindle checkpoint signaling, arguing that a traditional signal transduction cascade is integral to spindle checkpoint signaling.     

A new view of the spindle checkpoint

Previous studies of the spindle checkpoint suggested that its ability to prevent entry into anaphase was mediated by the inhibition of the anaphase-promoting complex (APC) ubiquitin ligase by Mad2. Two new studies challenge that view by demonstrating that another checkpoint protein, BubR1, is a far more potent inhibitor of APC function.     

Releasing the spindle assembly checkpoint without tension

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim''s activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim''s proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.     

Kinetochore stretching inactivates the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) monitors the attachment of microtubules to the kinetochore and inhibits anaphase when microtubule binding is incomplete. The SAC might also respond to tension; however, how cells can sense tension and whether its detection is important to satisfy the SAC remain controversial. We generated a HeLa cell line in which two components of the kinetochore, centromere protein A and Mis12, are labeled with green and red fluorophores, respectively. Live cell imaging of these cells reveals repetitive cycles of kinetochore extension and recoiling after biorientation. Under conditions in which kinetochore stretching is suppressed, cells fail to silence the SAC and enter anaphase after a delay, regardless of centromere stretching. Monitoring cyclin B levels as a readout for anaphase-promoting complex/cyclosome activity, we find that suppression of kinetochore stretching delays and decelerates cyclin B degradation. These observations suggest that the SAC monitors stretching of kinetochores rather than centromeres and that kinetochore stretching promotes silencing of the SAC signal.     

Deficient spindle assembly checkpoint in multiple myeloma

Multiple myeloma (MM) is a hematological disease characterized by an abnormal accumulation of plasma cells in the bone marrow. These cells have frequent cytogenetic abnormalities including translocations of the immunoglobulin heavy chain gene and chromosomal gains and losses. In fact, a singular characteristic differentiating MM from other hematological malignancies is the presence of a high degree of aneuploidies. As chromosomal abnormalities can be generated by alterations in the spindle assembly checkpoint (SAC), the functionality of such checkpoint was tested in MM. When SAC components were analyzed in MM cell lines, the RNA levels of most of them were conserved. Nevertheless, the protein content of some key constituents was very low in several cell lines, as was the case of MAD2 or CDC20 in RPMI-8226 or RPMI-LR5 cells. The recovery of their cellular content did not substantially affect cell growth, but improved their ability to segregate chromosomes. Finally, SAC functionality was tested by challenging cells with agents disrupting microtubule dynamics. Most of the cell lines analyzed exhibited functional defects in this checkpoint. Based on the data obtained, alterations both in SAC components and their functionality have been detected in MM, pointing to this pathway as a potential target in MM treatment.     

Mitosis: short-circuiting spindle checkpoint signaling

The spindle checkpoint forms an intricate signaling circuit to sense unattached kinetochores, to inhibit the anaphase-promoting complex/cyclosome (APC/C), and to delay anaphase onset. Using clever genetic experiments in the budding yeast, Lau and Murray define the endpoint of checkpoint signaling and provide key mechanistic insights into checkpoint inhibition of APC/C.     

Bub1-mediated adaptation of the spindle checkpoint

During cell division, the spindle checkpoint ensures accurate chromosome segregation by monitoring the kinetochore-microtubule interaction and delaying the onset of anaphase until each pair of sister chromosomes is properly attached to microtubules. The spindle checkpoint is deactivated as chromosomes start moving toward the spindles in anaphase, but the mechanisms by which this deactivation and adaptation to prolonged mitotic arrest occur remain obscure. Our results strongly suggest that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for the degradation of Bub1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest.     

Regulation of APC-Cdc20 by the spindle checkpoint

The spindle checkpoint ensures the fidelity of chromosome segregation in mitosis and meiosis. In response to defects in the mitotic apparatus, it blocks the activity of the anaphase-promoting complex, a large ubiquitin ligase required for chromosome segregation. Recent studies indicate that the spindle checkpoint monitors both the attachment of chromosomes to the mitotic spindle and the tension across the sister chromatid generated by microtubules. Upon checkpoint activation, checkpoint protein complexes containing BubR1(Mad3), Bub3, Mad2 and Cdc20 directly bind to the anaphase-promoting complex and inhibit its ligase activity. Therefore, the checkpoint proteins form a complex intracellular signalling network to inhibit the anaphase-promoting complex.     

Re-evaluating the role of Tao1 in the spindle checkpoint

The spindle checkpoint restrains anaphase onset and mitotic exit until all chromosomes are stably attached to the mitotic spindle via their kinetochores. The Tao1 protein kinase was recently reported as a novel spindle checkpoint component. When an siRNA was used to repress Tao1, the essential spindle checkpoint component Mad2 failed to localise to kinetochores, and cells rapidly exited mitosis. Tao1 was also shown to interact with BubR1, another essential checkpoint component, and be rapidly degraded after mitosis, a feature typical of many mitotic regulators. Here, we identify four different siRNAs that repress Tao1 protein levels as efficiently as the previously reported siRNA. However, these siRNAs do not override the spindle checkpoint. We also present data indicating that Tao1 does not interact with BubR1 and that it is not rapidly degraded after mitosis. We show that the previously reported siRNA not only represses Tao1 but also dramatically reduces Mad2 protein levels. Crucially, expression of exogenous Mad2, but not Tao1, rescued the spindle checkpoint phenotype induced by this siRNA. Thus, the key functional data implicating Tao1 in the spindle checkpoint can be explained by an off-target siRNA phenomenon that results in Mad2 inhibition. Taken together, our data do not support the notion that Tao1 is a component of the spindle checkpoint.     

A dynamical model of the spindle position checkpoint

The orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. In budding yeast, a surveillance mechanism called the spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother‐to‐daughter polarity axis. SPOC arrest relies upon inhibition of the GTPase Tem1 by the GTPase‐activating protein (GAP) complex Bfa1–Bub2. Importantly, reactions signaling mitotic exit take place at yeast centrosomes (named spindle pole bodies, SPBs) and the GAP complex also promotes SPB localization of Tem1. Yet, whether the regulation of Tem1 by Bfa1–Bub2 takes place only at the SPBs remains elusive. Here, we present a quantitative analysis of Bfa1–Bub2 and Tem1 localization at the SPBs. Based on the measured SPB‐bound protein levels, we introduce a dynamical model of the SPOC that describes the regulation of Bfa1 and Tem1. Our model suggests that Bfa1 interacts with Tem1 in the cytoplasm as well as at the SPBs to provide efficient Tem1 inhibition.     

In-silico modeling of the mitotic spindle assembly checkpoint

Background

The Mitotic Spindle Assembly Checkpoint (^MSAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer.

Principle Findings

We have constructed and validated for the human ^MSAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the ^MSAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the “Dissociation” and the “Convey” model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments.

Conclusion

Only in the controlled case, our models show ^MSAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for ^MSAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.