The Mitotic Checkpoint Complex controls the association of Cdc20 regulatory protein with the ubiquitin ligase APC/C in mitosis.

The ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) and its regulatory protein Cdc20 play important roles in the control of different stages of mitosis. APC/C associated with Cdc20 is active and promotes metaphase-anaphase transition by targeting for degradation inhibitors of anaphase initiation. Earlier in mitosis, premature action of APC/C is prevented by the mitotic checkpoint (or spindle assembly checkpoint) system, which ensures that anaphase is not initiated until all chromosomes are properly attached to the mitotic spindle. The active mitotic checkpoint system promotes the assembly of a Mitotic Checkpoint Complex (MCC), which binds to APC/C and inhibits its activity. The interaction of MCC with APC/C is strongly enhanced by Cdc20 bound to APC/C. While the association of Cdc20 with APC/C was known to be essential for both these stages of mitosis, it was not known how Cdc20 remains bound in spite of ongoing processes, phosphorylation and ubiquitylation, that stimulate its release from APC/C. We find that MCC strongly inhibits the release of Cdc20 from APC/C by the action of mitotic protein kinase Cdk1-cyclin B. This is not due to protection from phosphorylation of specific sites in Cdc20 that affect its interaction with APC/C. Rather, MCC stabilizes the binding to APC/C of partially phosphorylated forms of Cdc20. MCC also inhibits the autoubiquitylation of APC/C-bound Cdc20 and its ubiquitylation-promoted release from APC/C. We propose that these actions of MCC to maintain Cdc20 bound to APC/C in mitosis are essential for the control of mitosis during active mitotic checkpoint and in subsequent anaphase initiation.

Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes.

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.

CCDC68 Maintains Mitotic Checkpoint Activation by Promoting CDC20 Integration into the MCC.

The spindle assembly checkpoint (SAC) ensures chromosome segregation fidelity by manipulating unattached kinetochore-dependent assembly of the mitotic checkpoint complex (MCC). The MCC binds to and inhibits the anaphase promoting complex/cyclosome (APC/C) to postpone mitotic exit. However, the mechanism by which unattached kinetochores mediate MCC formation is not yet fully understood. Here, it is shown that CCDC68 is an outer kinetochore protein that preferentially localizes to unattached kinetochores. Furthermore, CCDC68 interacts with the SAC factor CDC20 to inhibit its autoubiquitination and MCC disassembly. Therefore, CCDC68 restrains APC/C activation to ensure a robust SAC and allow sufficient time for chromosome alignment, thus ensuring chromosomal stability. Hence, the study reveals that CCDC68 is required for CDC20-dependent MCC stabilization to maintain mitotic checkpoint activation.

Distinct roles of spindle checkpoint proteins in meiosis.

Gametes are produced via meiosis, a specialized cell division associated with frequent errors that cause birth defects and infertility. Uniquely in meiosis I, homologous chromosomes segregate to opposite poles, usually requiring their linkage by chiasmata, the products of crossover recombination.1 The spindle checkpoint delays cell-cycle progression until all chromosomes are properly attached to microtubules,2 but the steps leading to the capture and alignment of chromosomes on the meiosis I spindle remain poorly understood. In budding yeast meiosis I, Mad2 and Mad3BUBR1 are equally important for spindle checkpoint delay, but biorientation of homologs on the meiosis I spindle requires Mad2, but not Mad3BUBR1.3,4 Here we reveal the distinct functions of Mad2 and Mad3BUBR1 in meiosis I chromosome segregation. Mad2 promotes the prophase to metaphase I transition, while Mad3BUBR1 associates with the TOGL1 domain of Stu1CLASP, a conserved plus-end microtubule protein that is important for chromosome capture onto the spindle. Homologous chromosome pairs that are proficient in crossover formation but fail to biorient rely on Mad3BUBR1-Stu1CLASP to ensure their efficient attachment to microtubules and segregation during meiosis I. Furthermore, we show that Mad3BUBR1-Stu1CLASP are essential to rescue the segregation of mini-chromosomes lacking crossovers. Our findings define a new pathway ensuring microtubule-dependent chromosome capture and demonstrate that spindle checkpoint proteins safeguard the fidelity of chromosome segregation both by actively promoting chromosome alignment and by delaying cell-cycle progression until this has occurred.

The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction.

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of Drosophila melanogaster KNL1 (Spc105) has never been shown to bind MTs or to recruit PP1. Furthermore, the phosphoregulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell-based assays. A phosphoregulatory circuit that utilizes Aurora B kinase promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag and deletion/chimera mutants are used to define the interplay of MT and PP1 binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction.

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes.

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V-/- oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V-/- oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V-/- oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

Protein phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae.

Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the spindle position checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5, and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.

Inhibition of CDK4/6 kinases causes production of aneuploid oocytes by inactivating the spindle assembly checkpoint and accelerating first meiotic progression.

Cyclin D-CDK4/6 complex mediates the transition from the G1 to S phase in mammalian somatic cells. Meiotic oocytes pass through the G2/M transition and complete the first meiosis to reach maturation at the metaphase of meiosis II without intervening S phase, while Cyclin D-CDK4/6 complex is found to express during meiotic progression. Whether Cyclin D-CDK4/6 complex regulates meiotic cell cycle progression is not known. Here, we found its different role in oocyte meiosis: Cyclin D-CDK4/6 complex served as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Inhibition of CDK4/6 kinases disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, but unexpectedly accelerated meiotic progression by inactivating SAC, consequently resulting in production of aneuploid oocytes. Further studies showed that the MPF activity decrease before first polar body extrusion was accelerated probably by inactivation of the SAC to promote ubiquitin-mediated cyclin B1 degradation. Taken together, these data reveal a novel role of Cyclin D-CDK4/6 complex in mediating control of the SAC in female meiosis I.

Coupling of Cdc20 inhibition and activation by BubR1.

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.

WAPL orchestrates porcine oocyte meiotic progression via control of spindle assembly checkpoint activity.

BACKGROUND: In mitotic cells, WAPL acts as a cohesin release factor to remove cohesin complexes from chromosome arms during prophase to allow the accurate chromosome segregation in anaphase. However, we have recently documented that Wapl exerts a unique meiotic function in the spindle assembly checkpoint (SAC) control through maintaining Bub3 stability during mouse oocyte meiosis I. Whether this noncanonical function is conserved among species is still unknown. METHODS: We applied RNAi-based gene silencing approach to deplete WAPL in porcine oocytes, validating the conserved roles of WAPL in the regulation of SAC activity during mammalian oocyte maturation. We also employed immunostaining, immunoblotting and image quantification analyses to test the WAPL depletion on the meiotic progression, spindle assembly, chromosome alignment and dynamics of SAC protein in porcine oocytes. RESULTS: We showed that depletion of WAPL resulted in the accelerated meiotic progression by displaying the precocious polar body extrusion and compromised spindle assembly and chromosome alignment. Notably, we observed that the protein level of BUB3 was substantially reduced in WAPL-depleted oocytes, especially at kinetochores. CONCLUSIONS: Collectively, our data demonstrate that WAPL participates in the porcine oocyte meiotic progression through maintenance of BUB3 protein levels and SAC activity. This meiotic function of WAPL in oocytes is highly conserved between pigs and mice.

Role of Aurora B and Haspin kinases in the metaphase Topoisomerase II checkpoint.

DNA Topoisomerase II (TopoII) uses ATP hydrolysis to decatenate chromosomes so that sister chromatids can faithfully segregate in mitosis. When the TopoII enzyme cycle stalls due to failed ATP hydrolysis, the onset of anaphase is delayed, presumably to allow extra time for decatenation to be completed. Recent evidence revealed that, unlike the spindle assembly checkpoint, this TopoII checkpoint response requires Aurora B and Haspin kinases and is triggered by SUMOylation of the C-terminal domain of TopoII.

Mps1 controls spindle assembly, SAC, and DNA repair in the first cleavage of mouse early embryos.

Monopolar spindle-1 (Mps1) is a critical interphase regulator that also involves into the spindle assembly checkpoint for the cell cycle control in both mitosis and meiosis. However, the functions of Mps1 during mouse early embryo development is still unclear. In this study, we reported the important roles of Mps1 in the first cleavage of mouse embryos. Our data indicated that the loss of Mps1 activity caused precocious cleavage of zygotes to 2-cell embryos; however, prolonged culture disturbed the early embryo development to the blastocyst. We found that the spindle organization was disrupted after Mps1 inhibition, and the chromosomes were misaligned in the first cleavage. Moreover, the kinetochore-microtubule attachment was lost and Aurora B failed to accumulate to the kinetochores, indicating that the spindle assembly checkpoint (SAC) was activated. Furthermore, the inhibition of Mps1 activity resulted in an increase of DNA damage, which further induced oxidative stress, showing with positive γ-H2A.X signal and increased reactive oxygen species level. Ultimately, irreparable DNA damage and oxidative stress-activated apoptosis and autophagy, which was confirmed by the positive Annexin-V signal and increased autophagosomes. Taken together, our data indicated that Mps1 played important roles in the control of SAC and DNA repair during mouse early embryo development.

Unifying the mechanism of mitotic exit control in a spatiotemporal logical model.

The transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatiotemporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively; however, a unified understanding of how localisation and protein activity function together as a system is lacking. In this paper, we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. We show that our model is capable of correctly predicting the phenotype of the majority of mutants we tested, including mutants that cause proteins to mislocalise. We use a continuous time implementation of the model to demonstrate that Cdc14 Early Anaphase Release (FEAR) ensures robust timing of anaphase, and we verify our findings in living cells. Furthermore, we show that our model can represent measured cell-cell variation in Spindle Position Checkpoint (SPoC) mutants. This work suggests a general approach to incorporate spatial effects into logical models. We anticipate that the model itself will be an important resource to experimental researchers, providing a rigorous platform to test hypotheses about regulation of mitotic exit.

BUBR1 Pseudokinase Domain Promotes Kinetochore PP2A-B56 Recruitment, Spindle Checkpoint Silencing, and Chromosome Alignment.

The balance of phospho-signaling at the outer kinetochore is critical for forming accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. A major player in determining this balance is the PP2A-B56 phosphatase, which is recruited to the kinase attachment regulatory domain (KARD) of budding uninhibited by benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This unleashes a rapid, switch-like phosphatase relay that reverses mitotic phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase. Here, we demonstrate that the C-terminal pseudokinase domain of human BUBR1 is required to promote KARD phosphorylation. Mutation or removal of the pseudokinase domain results in decreased PP2A-B56 recruitment to the outer kinetochore attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. Our data, therefore, elucidate a function for the BUBR1 pseudokinase domain in ensuring accurate and timely exit from mitosis.

Homeostatic Control of Meiotic Prophase Checkpoint Function by Pch2 and Hop1.

Checkpoint cascades link cell cycle progression with essential chromosomal processes. During meiotic prophase, recombination and chromosome synapsis are monitored by what are considered distinct checkpoints. In budding yeast, cells that lack the AAA+ ATPase Pch2 show an impaired cell cycle arrest in response to synapsis defects. However, unperturbed pch2Δ cells are delayed in meiotic prophase, suggesting paradoxical roles for Pch2 in cell cycle progression. Here, we provide insight into the checkpoint roles of Pch2 and its connection to Hop1, a HORMA domain-containing client protein. Contrary to current understanding, we find that Pch2 (together with Hop1) is crucial for checkpoint function in response to both recombination and synapsis defects, thus revealing a shared meiotic checkpoint cascade. Meiotic checkpoint responses are transduced by DNA break-dependent phosphorylation of Hop1. Based on our data and on the described effect of Pch2 on HORMA topology, we propose that Pch2 promotes checkpoint proficiency by catalyzing the availability of signaling-competent Hop1. Conversely, we demonstrate that Pch2 can act as a checkpoint silencer, also in the face of persistent DNA repair defects. We establish a framework in which Pch2 and Hop1 form a homeostatic module that governs general meiotic checkpoint function. We show that this module can-depending on the cellular context-fuel or extinguish meiotic checkpoint function, which explains the contradictory roles of Pch2 in cell cycle control. Within the meiotic prophase checkpoint, the Pch2-Hop1 module thus operates analogous to the Pch2/TRIP13-Mad2 module in the spindle assembly checkpoint that monitors chromosome segregation.

A Biosensor for the Mitotic Kinase MPS1 Reveals Spatiotemporal Activity Dynamics and Regulation.

Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1-3]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3, 4]. To understand when and where MPS1 activation occurs and how MPS1 signaling is modulated during mitosis, we developed MPS1sen, a sensitive and specific FRET-based biosensor for MPS1 activity. By placing MPS1sen at different subcellular locations, we show that MPS1 activity initiates in the nucleus ∼9-12 min prior to nuclear envelope breakdown (NEB) in a kinetochore-dependent manner and reaches the cytoplasm at the start of NEB. Soon after initiation, MPS1 activity increases with switch-like kinetics, peaking at completion of NEB. We further show that timing and extent of pre-NEB MPS1 activity is regulated by Aurora B and PP2A-B56. MPS1sen phosphorylation declines in prometaphase as a result of formation of kinetochore-microtubule attachments, reaching low but still detectable levels at metaphase. Finally, leveraging the sensitivity and dynamic range of MPS1sen, we show deregulated MPS1 signaling dynamics in colorectal cancer cell lines and tumor organoids with diverse genomic instability phenotypes.

Vitrification-induced activation of lysosomal cathepsin B perturbs spindle assembly checkpoint function in mouse oocytes.

As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus-oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.

The Borealin dimerization domain interacts with Sgo1 to drive Aurora B-mediated spindle assembly.

The chromosomal passenger complex (CPC), which includes the kinase Aurora B, is a master regulator of meiotic and mitotic processes that ensure the equal segregation of chromosomes. Sgo1 is thought to play a major role in the recruitment of the CPC to chromosomes, but the molecular mechanism and contribution of Sgo1-dependent CPC recruitment is currently unclear. Using Xenopus egg extracts and biochemical reconstitution, we found that Sgo1 interacts directly with the dimerization domain of the CPC subunit Borealin. Borealin and the PP2A phosphatase complex can bind simultaneously to the coiled-coil domain of Sgo1, suggesting that Sgo1 can integrate Aurora B and PP2A activities to modulate Aurora B substrate phosphorylation. A Borealin mutant that specifically disrupts the Sgo1-Borealin interaction results in defects in CPC chromosomal recruitment and Aurora B-dependent spindle assembly, but not in spindle assembly checkpoint signaling at unattached kinetochores. These findings establish a direct molecular connection between Sgo1 and the CPC and have major implications for the different functions of Aurora B, which promote the proper interaction between spindle microtubules and chromosomes.

The conserved AAA-ATPase PCH-2 TRIP13 regulates spindle checkpoint strength.

Spindle checkpoint strength is dictated by the number of unattached kinetochores, cell volume, and cell fate. We show that the conserved AAA-ATPase PCH-2/TRIP13, which remodels the checkpoint effector Mad2 from an active conformation to an inactive one, controls checkpoint strength in Caenorhabditis elegans. Having previously established that this function is required for spindle checkpoint activation, we demonstrate that in cells genetically manipulated to decrease in cell volume, PCH-2 is no longer required for the spindle checkpoint or recruitment of Mad2 at unattached kinetochores. This role is not limited to large cells: the stronger checkpoint in germline precursor cells also depends on PCH-2. PCH-2 is enriched in germline precursor cells, and this enrichment relies on conserved factors that induce asymmetry in the early embryo. Finally, the stronger checkpoint in germline precursor cells is regulated by CMT-1, the ortholog of p31comet, which is required for both PCH-2's localization to unattached kinetochores and its enrichment in germline precursor cells. Thus, PCH-2, likely by regulating the availability of inactive Mad2 at and near unattached kinetochores, governs checkpoint strength. This requirement may be particularly relevant in oocytes and early embryos enlarged for developmental competence, cells that divide in syncytial tissues, and immortal germline cells.

The Spindle Assembly Checkpoint Functions during Early Development in Non-Chordate Embryos.

In eukaryotic cells, a spindle assembly checkpoint (SAC) ensures accurate chromosome segregation, by monitoring proper attachment of chromosomes to spindle microtubules and delaying mitotic progression if connections are erroneous or absent. The SAC is thought to be relaxed during early embryonic development. Here, we evaluate the checkpoint response to lack of kinetochore-spindle microtubule interactions in early embryos of diverse animal species. Our analysis shows that there are two classes of embryos, either proficient or deficient for SAC activation during cleavage. Sea urchins, mussels, and jellyfish embryos show a prolonged delay in mitotic progression in the absence of spindle microtubules from the first cleavage division, while ascidian and amphioxus embryos, like those of Xenopus and zebrafish, continue mitotic cycling without delay. SAC competence during early development shows no correlation with cell size, chromosome number, or kinetochore to cell volume ratio. We show that SAC proteins Mad1, Mad2, and Mps1 lack the ability to recognize unattached kinetochores in ascidian embryos, indicating that SAC signaling is not diluted but rather actively silenced during early chordate development.