Defects In Mitotic Exit Research Articles

The budding yeast Polo-like kinase Cdc5 is released from the nucleus during anaphase for timely mitotic exit

Polo-like kinases are important regulators of multiple mitotic events; however, how Polo-like kinases are spatially and temporally regulated to perform their many tasks is not well understood. Here, we examined the subcellular localization of the budding yeast Polo-like kinase Cdc5 using a functional Cdc5-GFP protein expressed from the endogenous locus. In addition to the well-described localization of Cdc5 at the spindle pole bodies (SPBs) and the bud neck, we found that Cdc5-GFP accumulates in the nucleus in early mitosis but is released to the cytoplasm in late mitosis in a manner dependent on the Cdc14 phosphatase. This Cdc5 release from the nucleus is important for mitotic exit because artificial sequestration of Cdc5 in the nucleus by addition of a strong nuclear localization signal (NLS) resulted in mitotic exit defects. We identified a key cytoplasmic target of Cdc5 as Bfa1, an inhibitor of mitotic exit. Our study revealed a novel layer of Cdc5 regulation and suggests the existence of a possible coordination between Cdc5 and Cdc14 activity.

Alterations to O‐GlcNAc cycling disrupt mitotic phosphorylation (555.16)

O‐GlcNAc is a post‐translational modification (PTM) consisting of a single N‐acetylglucosamine moiety attached to serine and threonine residues in nuclear and cytoplasmic proteins. O‐GlcNAc levels can cycle dynamically in response to hormones, nutrients, or stress in order to regulate a multitude of cellular processes. For instance O‐GlcNAc cycling is an important regulator of mitosis. Recently, we demonstrated that cells with a gain of function in either O‐GlcNAc transferase (OGT) or O‐GlcNAcase (OGA) have disrupted mitotic spindles and mitotic exit defects. OGT/OGA gain of function caused the formation of large poorly condensed spindle chromosomes, misaligned kinetochores, and an increase in multipolar spindles. This phenotype was caused partially by a disruption in phosphorylation. In this current study, our objective is to determine how alterations in O‐GlcNAc cycling disrupt mitotic phosphorylation cascades. We employed quantitative proteomics in order to identify changes to mitotic phosphorylation. We were able to quantitate both protein expression changes as well as alterations in PTM’s in OGT or OGA gain of function cells. We identified over 1000 proteins whose expression as well as PTM status was altered by aberrant O‐GlcNAcylation. These results demonstrate the cross‐talk between protein modifications involved in regulating mitotic progression and suggest that diseases that disrupt one PTM will also alter the actions of other PTM’s.Grant Funding Source: NIH‐NHLBI Contracts: HHSN268201000031C ‐ CPC; NIH Grants: P41 RR010888/GM104603 – MSR R21 HL107993 ;

Chromosomal breaks during mitotic catastrophe trigger γH2AX–ATM–p53-mediated apoptosis

Although the cause and outcome of mitotic catastrophe (MC) has been thoroughly investigated, precisely how the ensuing lethality is regulated during or following this process and what signals are involved remain unknown. Moreover, the mechanism of the decision of cell death modalities following MC is still not well characterised. We demonstrate here a crucial role of the γH2AX-ATM-p53 pathway in the regulation of the apoptotic outcome of MC resulting from cells entering mitosis with damaged DNA. In addition to p53 deficiency, the depletion of ATM (ataxia telangiectasia mutated), but not ATR (ataxia telangiectasia and Rad3-related protein), protected against apoptosis and shifted cell death towards necrosis. Activation of this pathway is triggered by the augmented chromosomal damage acquired during anaphase in doxorubicin-treated cells lacking 14-3-3σ (also known as epithelial cell marker protein-1 or stratifin). Moreover, cells that enter mitosis with damaged DNA encounter segregation problems because of their abnormal chromosomes, leading to defects in mitotic exit, and they therefore accumulate in G1 phase. These multi- or micronucleated cells are prevented from cycling again in a p53- and p21-dependent manner, and subsequently die. Because increased chromosomal damage resulting in extensive H2AX phosphorylation appears to be a direct cause of catastrophic mitosis, our results describe a mechanism that involves generation of additional DNA damage during MC to eliminate chromosomally unstable cells.

Cdc14b: A New Player in Aging

Cdc14 encodes a dual specificity protein phosphatase essential for mitotic exit. There are two Cdc14 isoforms (a and b) in vertebrates. Cdc14a localizes to the centrosome and Cdc14b localizes to the nucleolus [1]. The two mammalian Cdc14 isoforms have been speculated to exert essential mitotic functions, given the role of budding yeast ScCdc14 played in the cell cycle and the fact that both human CDC14A and CDC14B could rescue the mitotic exit defects of ScCdc14 mutants [2, 3]. However, it is apparent now that neither Cdc14a nor Cdc14b is required for mitotic exit, not even for the cell cycle [4-6]. Instead, both of them were shown to be involved in DNA damage response [4-7]. It is unclear how these two paralogous phosphatases are involved in DNA damage repair. It is imaginable that they are probably required to dephosphorylate some substrates that participate in the repair process. When Cdc14b-deficient mice were left to age, they develop cataracts and kyphosis at early ages [6], suggesting a role of Cdc14b in preventing the early onset of aging. Although this premature aging phenotype is consistent with the role of Cdc14b in DNA damage repair, we could not exclude the possibility that Cdc14b is involved in other cellular processes that are more important in preventing aging. Thus, we noticed a synergistic effect on aging when Cdc14b deficiency was combined with Cdh1 haploinsufficiency (Wei and Zhang, unpublished observation). Cdh1 is the adaptor protein for the E3 ubiquitin ligase APC (anaphase promoting complex). In budding yeasts Cdh1 is downstream from Cdc14 [8], which may also be true in mammals. However, there is no evidence suggesting a role of Cdh1 in DNA damage repair. Thus, it is possible that Cdc14b's function in aging is through Cdh1 in processes not related to damage repair. It will be interesting to see if Cdc14a also plays a role in aging.

Mutual regulation of cyclin-dependent kinase and the mitotic exit network

The mitotic exit network (MEN) is a spindle pole body (SPB)-associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1-Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2-Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1-Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.

Cyclin-Specific Control of Ribosomal DNA Segregation

Following chromosome duplication in S phase of the cell cycle, the sister chromatids are linked by cohesin. At the onset of anaphase, separase cleaves cohesin and thereby initiates sister chromatid separation. Separase activation results from the destruction of its inhibitor, securin, which is triggered by a ubiquitin ligase called the anaphase-promoting complex (APC). Here, we show in budding yeast that securin destruction and, thus, separase activation are not sufficient for the efficient segregation of the repetitive ribosomal DNA (rDNA). We find that rDNA segregation also requires the APC-mediated destruction of the S-phase cyclin Clb5, an activator of the protein kinase Cdk1. Mutations that prevent Clb5 destruction are lethal and cause defects in rDNA segregation and DNA synthesis. These defects are distinct from the mitotic-exit defects caused by stabilization of the mitotic cyclin Clb2, emphasizing the importance of cyclin specificity in the regulation of late-mitotic events. Efficient rDNA segregation, both in mitosis and meiosis, also requires APC-dependent destruction of Dbf4, an activator of the protein kinase Cdc7. We speculate that the dephosphorylation of Clb5-specific Cdk1 substrates and Dbf4-Cdc7 substrates drives the resolution of rDNA in early anaphase. The coincident destruction of securin, Clb5, and Dbf4 coordinates bulk chromosome segregation with segregation of rDNA.

Different Levels of Bfa1/Bub2 GAP Activity Are Required to Prevent Mitotic Exit of Budding Yeast Depending on the Type of Perturbations

In budding yeast, Tem1 is a key regulator of mitotic exit. Bfa1/Bub2 stimulates Tem1 GTPase activity as a GTPase-activating protein (GAP). Lte1 possesses a guanine-nucleotide exchange factor (GEF) domain likely for Tem1. However, recent observations showed that cells may control mitotic exit without either Lte1 or Bfa1/Bub2 GAP activity, obscuring how Tem1 is regulated. Here, we assayed BFA1 mutants with varying GAP activities for Tem1, showing for the first time that Bfa1/Bub2 GAP activity inhibits Tem1 in vivo. A decrease in GAP activity allowed cells to bypass mitotic exit defects. Interestingly, different levels of GAP activity were required to prevent mitotic exit depending on the type of perturbation. Although essential, more Bfa1/Bub2 GAP activity was needed for spindle damage than for DNA damage to fully activate the checkpoint. Conversely, Bfa1/Bub2 GAP activity was insufficient to delay mitotic exit in cells with misoriented spindles. Instead, decreased interaction of Bfa1 with Kin4 was observed in BFA1 mutant cells with a defective spindle position checkpoint. These findings demonstrate that there is a GAP-independent surveillance mechanism of Bfa1/Bub2, which, together with the GTP/GDP switch of Tem1, may be required for the genomic stability of cells with misaligned spindles.

Budding Yeast DNA Damage Adaptation Mutants Exhibit Defects in Mitotic Exit

In the presence of double strand breaks, DNA damage checkpoint halts cell cycle progression. However, cells ultimately escape the checkpoint arrest and re-enter cell cycle in the presence of irreparable DNA damage. cdc5-ad was identified as a mutant that fails to adapt to the cell cycle arrest induced by DNA damage checkpoint. In budding yeast, Cdc5 protein kinase is a component of both MEN and FEAR pathways that are required for mitotic exit. It remains unclear whether the adaptation defect of cdc5-ad mutant cells is related to the function of Cdc5 in mitotic exit. Here we present evidence indicating that cdc5-ad mutant cells exhibit defects in mitotic exit. cdc5-ad mutant cells are sensitive to high dosage of Amn1, a negative regulator of MEN. It also shows synthetic growth defects with mutants in MEN pathway. Moreover, mutants in FEAR pathway exhibit defects in DNA damage adaptation. Thus, we conclude that the compromised mitotic exit pathway contributes to DNA damage adaptation defects in cdc5-ad mutant cells.

Phosphatase 2A negatively regulates mitotic exit in Saccharomyces cerevisiae.

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.

Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast.

ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Δlte1 mutant and a Δras1 Δras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.