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Final Report Summary - ASYMMETRY IN MEN (Generation of asymmetry in mitotic exit network (MEN) signaling)

Executive Summary:

The cell cycle is an ordered series of events that allows for the duplication of a cell. This process is crucial to the development and growth of all living organisms. During the cell cycle, chromosomes are first replicated and then distributed between the progeny. Finally, and once that the duplicated genome has been equally partitioned, mitotic exit takes place. This final cell cycle transition involves, among other processes, the disassembly of the mitotic spindle (a bipolar array of microtubules that allows for the segregation of the chromosomes) and the completion of cytokinesis. Mitotic exit is determined by the inactivation of the cyclin-dependent kinases (CDKs), which are enzymes that drive cell cycle progression together with their regulatory cyclin subunits. In budding yeast, the main determinant of mitotic exit is the protein phosphatase Cdc14, whose activity is mainly regulated by changes in its localization. Cdc14 is sequestered in the nucleolus from G1 to metaphase, but it is released from this cellular structure at anaphase onset. Once released, Cdc14 determines the reversal of the phosphorylation events promoted by mitotic CDKs, which eventually determines their inactivation. The sustained release of Cdc14 and the successful completion of mitosis require the activity of the MEN (mitotic exit network), a Ras-like signaling cascade that is initiated by the Tem1 GTPase (Figure 1). The activity of Tem1 is inhibited until anaphase by the two-component GTPase-activating protein (GAP) Bfa1-Bub2. This GAP is maintained in an active state by the Kin4 kinase, which prevents the inhibitory phosphorylation of Bfa1 by the Polo-like kinase Cdc5. At anaphase onset, Cdc5 inhibits Bfa1-Bub2, while Lte1, the positive regulator of the MEN, simultaneously stimulates the activity of Tem1. Once Tem1 is active, MEN signaling is triggered and promotes the final release of Cdc14 and the subsequent inactivation of mitotic CDK activity.

Interestingly, the localization of most MEN components during the cell cycle is asymmetric (Figure 2). As such, Lte1 localizes in the bud cortex concomitantly with bud formation, while Kin4 localizes in the mother cell cortex and, briefly during anaphase, on the spindle pole body (SPB, the equivalent of the centrosome in yeasts) that stays in the mother cell. Finally, Tem1, Bfa1 and Bub2 form a complex (the so-called Tem1 complex), which is loaded onto the SPBs and also localizes asymmetrically. During anaphase, the Tem1 complex only loads onto the SPB that enters the daughter cell, but not on the one that remains in the mother cell. This asymmetric localization of MEN components is thought to allow the cell to generate MEN activating (the bud) and MEN inhibiting (the mother) zones, so that only when Tem1 (the protein that initiates the pathway) is in the same zone that Lte1 (a MEN activator) mitotic exit is allowed.

The cells count with a number of surveillance mechanisms to ensure that the replicated genomic material is protected from damage and correctly distributed between the daughter and mother cells during mitosis. In this way, chromosomal DNA damage triggers the DNA damage checkpoint (DDC), which blocks cell cycle progression to provide the cells with time to repair the genomic material before further progressing into mitosis. The cells also monitor the proper attachment of all sister kinetochores to the spindle in a bipolar fashion by means of the spindle assembly checkpoint (SAC), a surveillance mechanism that regulates the metaphase to anaphase transition. Finally, in cells that display some type of asymmetry during mitosis, as it is the case of budding yeast, it is also essential that the spindle be correctly positioned with respect to the division site, which is ensured by the spindle position checkpoint (SPOC). Remarkably, and despite the diverse signals that activate the DDC, the SAC and the SPOC, as well as the different cell cycle stages where these surveillance mechanisms are triggered, all previous checkpoints have been shown to inhibit mitotic exit.

The main goal of this project is to shed light into the mechanisms by which the asymmetric localization of the Tem1 complex is established and to analyze the consequences that disrupting this asymmetry have on cell cycle progression. We have also analyzed the role of the asymmetric localization of the Tem1 complex in the regulation of the checkpoints that ensure that the genome is faithfully distributed during mitosis. Finally, we have analyzed the alternative mechanisms that regulate the MEN after the activation of checkpoints that do not depend on this asymmetry for their functionality.

During the extension of this International Reintegration Grant, we have made important contributions into our understanding of the role that the localization of Tem1 has on the regulation of mitotic exit. In this sense, we have demonstrated that the localization of the Tem1 GTPase to the SPBs is an essential requirement in order to exit from mitosis. Interestingly, however, we have demonstrated that the constitutive loading of Tem1 does not affect cell cycle progression, even when the GTPase is symmetrically localized to both SPBs (Valerio-Santiago and Monje-Casas, JCB (2011); 192(4): 599-614). We have also analyzed how the localization of the Tem1 complex interferes with the functionality of the DDC, the SAC, and the SPOC, and found out that while the DDC and the SAC are not affected by the constitutive loading of Tem1, the SPOC is no longer functional under these circumstances (Valerio-Santiago and Monje-Casas, JCB (2011); 192(4): 599-614). This allowed us to establish the importance of mechanisms that regulate the MEN both upstream and downstream of Tem1 and that avoid a premature mitotic exit before anaphase onset. Additionally, we have proposed that the asymmetric localization of Tem1 is a consequence of the mechanisms that ensure that mitotic exit only takes place when one SPB has entered the daughter cell. Exclusion of Tem1 from the mother SPB is important to block MEN activation when the spindle is not properly aligned and both SPBs are located within the context of the mother cell. However, loading of Tem1 onto the SPBs in the mother cell does not pose a threat for cell viability under normal growth conditions because the mechanisms that position the spindle are highly efficient.

As previously indicated, we have demonstrated that there are mechanisms that restrain MEN activation even after DDC and SAC activation when Tem1 is constitutively loaded onto SPBs. Since little was known about how DNA damage triggers inhibition of mitotic exit, during the last period of this International Reintegration Grant we have also analyzed the mechanisms leading to inactivation of the MEN when the DDC is triggered. Our studies have allowed us to demonstrate that inhibition of the MEN is specifically required when telomeres are damaged, but not to face other types of chromosomal DNA damage (Valerio-Santiago et al., PLoS Genetics (2013); 9(10): e1003859). These results are in agreement with previous data in mammals suggesting the existence of a putative telomere-specific DNA damage response that inhibits mitotic exit. Furthermore, we have demonstrated that the mechanism by which MEN is inactivated when telomeres are damaged relies on Rad53, an important DDC effector, which inhibits Bfa1 phosphorylation by Cdc5. Therefore, our data suggest that a common theme for all the mitotic checkpoints that rely on the inactivation of the MEN is the inhibition of Bfa1 phosphorylation by Polo-kinase, and that the different surveillance mechanisms mainly diverge in the strategies by which this inhibition is achieved.

The regulation of the mitotic exit process is of pivotal importance for the cell. The core signaling module of MEN, constituted by a Ste20-like kinase (Cdc15), a MOB co-activator (Mob1), a kinase from the LATS/NDR kinase (Dbf2), and the Cdc14 phosphatase, is highly conserved from yeast to humans. Interestingly, deregulation of the Salvador-Hippo-Warts pathway, the homologue of the MEN in higher eukaryotes, has been associated with tumorigenesis in mammals. Advances in our knowledge about the regulation of mitotic exit will be therefore essential in our understanding of the mechanisms that underlay the carcinogenesis process. Cancer cells can resist treatment with antimitotic drugs that perturb spindle assembly and activate the SAC, like taxanes or vinca alkaloids, by prematurely exiting mitosis. An increasing body of evidences suggests that blocking mitotic exit progression may be a better therapeutic strategy than inhibiting earlier stages of the cell cycle with the previous drugs. Therefore, further understanding of the regulation of mitotic exit will also be beneficial in the future development of possible new cures for this disease.


Margarita Martínez-Pais Loscertales, (Chief of Research Services)
Tel.: +34 954 48 81 29
Fax: +34-954488117
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