Mutual antagonism between the anaphase promoting complex and the spindle assembly checkpoint contributes to mitotic timing in Caenorhabditis elegans

The anaphase promoting complex/cyclosome (APC/C) triggers the separation of sister chromatids and exit from mitosis across eukaryotic evolution. The APC/C is inhibited by the spindle assembly checkpoint (SAC) until all chromosomes have achieved bipolar attachment, but whether the APC/C reciprocally regulates the SAC is less understood. Here, we report the characterization of a novel allele of the APC5 component SUCH-1 in Caenorhabditis elegans. We find that some such-1(t1668) embryos lack paternally contributed DNA and centrioles and assemble a monopolar spindle in the one-cell stage. Importantly, we show that mitosis is drastically prolonged in these embryos, as well as in embryos that are otherwise compromised for APC/C function and assemble a monopolar spindle. This increased duration of mitosis is dependent on the SAC, since inactivation of the SAC components MDF-1/MAD1 or MDF-2/MAD2 rescues proper timing in these embryos. Moreover, partial depletion of the E1 enzyme uba-1 significantly increases mitosis duration upon monopolar spindle assembly. Taken together, our findings raise the possibility that the APC/C negatively regulates the SAC and, therefore, that the SAC and the APC/C have a mutual antagonistic relationship in C. elegans embryos.     

Suppressors of spindle checkpoint defect (such) mutants identify new mdf-1/MAD1 interactors in Caenorhabditis elegans

The spindle assembly checkpoint (SAC) governs the timing of metaphase-to-anaphase transition and is essential for genome stability. The Caenorhabditis elegans mutant strain gk2 carries a deletion within the mdf-1/MAD1 gene that results in death of the homozygous strain after two or three generations. Here we describe 11 suppressors of the mdf-1(gk2) lethality, 10 identified in an ethyl methanesulfonate (EMS) mutagenesis screen and 1 isolated using the dog-1(gk10) (deletions of guanine-rich DNA) mutator strain. Using time-lapse imaging of early embryonic cells and germline mitotic division, we demonstrate that there are two classes of suppressors. Eight suppressors compensate for the loss of the checkpoint by delaying mitotic progression, which coincides with securin (IFY-1/Pds1) accumulation; three suppressors have normal IFY-1/Pds1 levels and normal anaphase onset. Furthermore, in the class of suppressors with delayed mitotic progression, we have identified four alleles of known suppressors emb-30/APC4 and fzy-1/CDC20, which are components of the anaphase-promoting complex/cyclosome (APC/C). In addition, we have identified another APC/C component capable of bypassing the checkpoint requirement that has not previously been described in C. elegans. The such-1/APC5-like mutation, h1960, significantly delays anaphase onset both in germline and in early embryonic cells.     

Meiotic control of the APC/C: similarities & differences from mitosis

The anaphase promoting complex is a highly conserved E3 ligase complex that mediates the destruction of key regulatory proteins during both mitotic and meiotic divisions. In order to maintain ploidy, this destruction must occur after the regulatory proteins have executed their function. Thus, the regulation of APC/C activity itself is critical for maintaining ploidy during all types of cell divisions. During mitotic cell division, two conserved activator proteins called Cdc20 and Cdh1 are required for both APC/C activation and substrate selection. However, significantly less is known about how these proteins regulate APC/C activity during the specialized meiotic nuclear divisions. In addition, both budding yeast and flies utilize a third meiosis-specific activator. In Saccharomyces cerevisiae, this meiosis-specific activator is called Ama1. This review summarizes our knowledge of how Cdc20 and Ama1 coordinate APC/C activity to regulate the meiotic nuclear divisions in yeast.     

Multiple Requirements of PLK1 during Mouse Oocyte Maturation

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1’s functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.     

EMB-30: an APC4 homologue required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans

Here we show that emb-30 is required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. Germline-specific emb-30 mutant alleles block the meiotic divisions. Mutant oocytes, fertilized by wild-type sperm, set up a meiotic spindle but do not progress to anaphase I. As a result, polar bodies are not produced, pronuclei fail to form, and cytokinesis does not occur. Severe-reduction-of-function emb-30 alleles (class I alleles) result in zygotic sterility and lead to germline and somatic defects that are consistent with an essential role in promoting the metaphase-to-anaphase transition during mitosis. Analysis of the vulval cell lineages in these emb-30(class I) mutant animals suggests that mitosis is lengthened and eventually arrested when maternally contributed emb-30 becomes limiting. By further reducing maternal emb-30 function contributed to class I mutant animals, we show that emb-30 is required for the metaphase-to-anaphase transition in many, if not all, cells. Metaphase arrest in emb-30 mutants is not due to activation of the spindle assembly checkpoint but rather reflects an essential emb-30 requirement for M-phase progression. A reduction in emb-30 activity can suppress the lethality and sterility caused by a null mutation in mdf-1, a component of the spindle assembly checkpoint machinery. This result suggests that delaying anaphase onset can bypass the spindle checkpoint requirement for normal development. Positional cloning established that emb-30 encodes the likely C. elegans orthologue of APC4/Lid1, a component of the anaphase-promoting complex/cyclosome, required for the metaphase-to-anaphase transition. Thus, the anaphase-promoting complex/cyclosome is likely to be required for all metaphase-to-anaphase transitions in a multicellular organism.     

Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/C(Cdc20)), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.     

A novel destruction sequence targets the meiotic regulator Spo13 for anaphase-promoting complex-dependent degradation in anaphase I

The anaphase-promoting complex (APC) or cyclosome is a multisubunit ubiquitin-protein ligase that ubiquitinates and thereby promotes the destruction of the mitotic cyclins and the separase inhibitor, securin. The contributions of the APC to progression through the meiotic program are not clear. To clarify the function of the APC in meiosis, we screened several yeast meiotic proteins as APC substrates in vitro. We found that the meiotic regulator Spo13 is an APC substrate that is degraded during anaphase I. Spo13 is expressed only in meiotic cells, where it has multiple functions, including the promotion of monopolar chromosome attachment in the first division. Spo13 ubiquitination by the APC depends on an LxExxxN sequence (residues 26-32) that is distinct from previously described destruction sequences of APC substrates. Mutation of one residue, leucine 26, prevented Spo13 ubiquitination by the APC in vitro and stabilized the protein through the meiotic divisions. Analysis of meiotic progression and spore viability of yeast containing the stabilized Spo13 mutant revealed no significant defects, indicating that Spo13 destruction in anaphase I is not essential for meiosis. We propose that Spo13 destruction is one of multiple mechanisms underlying the switch from monopolar to bipolar chromosome attachment between the meiotic divisions.     

Requirement for PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase is lessened by the dosage increase of a novel gene sds23+.

Ubiquitin-dependent proteolysis is required for the onset of anaphase. We show that protein dephosphorylation by protein phosphatase 1 (PP1) is also essential for initiating anaphase in fission yeast. PP1 may directly or indirectly regulate the 20S cyclosome/APC (anaphase-promoting complex) required for anaphase-promoting proteolysis. Using anti-phosphopeptide antibodies, PP1 is shown to be dephosphorylated at the C-terminus, upon the onset of anaphase, for reactivation. sds23+, a novel gene, is a multicopy suppressor for mutations in PP1 and the 20S cyclosome/APC, implying that the gene dosage increase can relieve the requirement for PP1 and the cyclosome/APC for the onset of anaphase. The sds23+ gene is not essential for cell viability, but a mutant with the gene deleted cannot form colonies at 22 and 36 degrees C. In the sds23 deletion mutant, the progression of anaphase and cytokinesis is retarded and cell shape is aberrant. These defects are overcome by plasmids carrying the genes encoding subunits of the 20S cyclosome/APC or PP1. These results demonstrate functions other than promoting anaphase for the components of the 20S cyclosome/APC and also a close functional relationship of Sds23 with PP1 and 20S cyclosome/APC.     

Regulation of MBK-2/Dyrk kinase by dynamic cortical anchoring during the oocyte-to-zygote transition

Background: Successful transition from oocyte to zygote depends on the timely degradation of oocyte proteins to prepare for embryonic development. In C. elegans, degradation of the oocyte protein MEI-1 depends on MBK-2, a kinase that phosphorylates MEI-1 shortly after fertilization during the second meiotic division. Results: Here we report that precise timing of MEI-1 phosphorylation depends on the cell cycle-regulated release of MBK-2 from the cortex. Prior to the meiotic divisions, MBK-2 is tethered at the cortex by EGG-3, an oocyte protein required for egg activation (see [1], accompanying paper in this issue). During the meiotic divisions, EGG-3 is internalized and degraded in an APC/C (anaphase-promoting complex/cyclosome)-dependent manner. EGG-3 internalization and degradation correlate with MBK-2 release from the cortex and MEI-1 phosphorylation in the cytoplasm. In an egg-3 mutant, MEI-1 is phosphorylated and degraded prematurely. Conclusion: We suggest that successful transition from an oocyte to a zygote depends on the cell cycle-regulated relocalization of key regulators from the cortex to the cytoplasm of the egg.     

Anaphase Promoting Complex or Cyclosome?

The anaphase promoting complex/cyclosome (APC/C) was initially described as a multi-subunit protein complex that ubiquitinates anaphase inhibitors thus targeting them for destruction by proteasomes to initiate loss of sister chromatid cohesion. However, recent studies have identified important new functions of the APC/C. Moreover, sister centromere separation can occur in the absence of APC/C activity in mammals, indicating that anaphase onset might be triggered by multiple factors. Here we discuss whether the APC/C functions primarily as the anaphase trigger, or whether it has more general properties, relevant for cell cycle control at multiple developmental and cell cycle stages. Additionally, we discuss the validity of the APC-dependent model for sister segregation in mammals.     

The spindle and kinetochore–associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.     

The Cdc20 (Fzy)/Cdh1-related protein, Cort, cooperates with Fzy in cyclin destruction and anaphase progression in meiosis I and II in Drosophila

Meiosis is a highly specialized cell division that requires significant reorganization of the canonical cell-cycle machinery and the use of meiosis-specific cell-cycle regulators. The anaphase-promoting complex (APC) and a conserved APC adaptor, Cdc20 (also known as Fzy), are required for anaphase progression in mitotic cells. The APC has also been implicated in meiosis, although it is not yet understood how it mediates these non-canonical divisions. Cortex (Cort) is a diverged Fzy homologue that is expressed in the female germline of Drosophila, where it functions with the Cdk1-interacting protein Cks30A to drive anaphase in meiosis II. Here, we show that Cort functions together with the canonical mitotic APC adaptor Fzy to target the three mitotic cyclins (A, B and B3) for destruction in the egg and drive anaphase progression in both meiotic divisions. In addition to controlling cyclin destruction globally in the egg, Cort and Fzy appear to both be required for the local destruction of cyclin B on spindles. We find that cyclin B associates with spindle microtubules throughout meiosis I and meiosis II, and dissociates from the meiotic spindle in anaphase II. Fzy and Cort are required for this loss of cyclin B from the meiotic spindle. Our results lead to a model in which the germline-specific APC(Cort) cooperates with the more general APC(Fzy), both locally on the meiotic spindle and globally in the egg cytoplasm, to target cyclins for destruction and drive progression through the two meiotic divisions.     

emb-1 encodes the APC16 subunit of the Caenorhabditis elegans anaphase-promoting complex

In the nematode Caenorhabditis elegans, temperature-sensitive mutants of emb-1 arrest as one-cell embryos in metaphase of meiosis I in a manner that is indistinguishable from embryos that have been depleted of known subunits of the anaphase-promoting complex or cyclosome (APC/C). Here we show that the emb-1 phenotype is enhanced in double mutant combinations with known APC/C subunits and suppressed in double mutant combinations with known APC/C suppressors. In addition to its meiotic function, emb-1 is required for mitotic proliferation of the germline. These studies reveal that emb-1 encodes K10D2.4, a homolog of the small, recently discovered APC/C subunit, APC16.     

The anaphase promoting complex/cyclosome: a machine designed to destroy

The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase that has essential functions in and outside the eukaryotic cell cycle. It is the most complex molecular machine that is known to catalyse ubiquitylation reactions, and it contains more than a dozen subunits that assemble into a large 1.5-MDa complex. Recent discoveries have revealed an unexpected multitude of mechanisms that control APC/C activity, and have provided a first insight into how this unusual ubiquitin ligase recognizes its substrates.     

A novel function of anaphase promoting complex subunit 10 in tumor progression in non-small cell lung cancer

The anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase, is responsible for the transition from metaphase to anaphase and the exit from mitosis. The anaphase promoting complex subunit 10 (APC10), a subunit of the APC/C, executes a vital function in substrate recognition. However, no research has reported the connection between APC10 and cancer until now. In this study, we uncovered a novel, unprecedented role of APC10 in tumor progression, which is independent of APC/C. First, aberrant increase of APC10 expression was validated in non-small cell lung cancer (NSCLC) cells and tissues, and the absence of APC10 repressed cell proliferation and migration. Of great interest, we found that APC10 inhibition induced cell cycle arrest at the G0/G1 phase and reduced the expression of the APC/C substrate, Cyclin B1; this finding is different from the conventional concept of the accumulation of Cyclin B1 and cell cycle arrest in metaphase. Further, APC10 was found to interact with glutaminase C (GAC), and the inhibition of APC10 weakened glutamine metabolism and induced excessive autophagy. Taken together, these findings identify a novel function of APC10 in the regulation of NSCLC tumorigenesis and point to the possibility of APC10 as a new target for cancer therapy.     

Cdk1 promotes kinetochore bi-orientation and regulates Cdc20 expression during recovery from spindle checkpoint arrest

The spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance pathway, prevents chromosome segregation in response to conditions that disrupt the kinetochore-microtubule attachment. Removal of the checkpoint-activating stimulus initiates recovery during which spindle integrity is restored, kinetochores become bi-oriented, and cells initiate anaphase. Whether recovery ensues passively after the removal of checkpoint stimulus, or requires mediation by specific effectors remains uncertain. Here, we report two unrecognized functions of yeast Cdk1 required for efficient recovery from SAC-induced arrest. We show that Cdk1 promotes kinetochore bi-orientation during recovery by restraining premature spindle elongation thereby extinguishing SAC signalling. Moreover, Cdk1 is essential for sustaining the expression of Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) required for anaphase progression. We suggest a model in which Cdk1 activity promotes recovery from SAC-induced mitotic arrest by regulating bi-orientation and APC/C activity. Our findings provide fresh insights into the regulation of mitosis and have implications for the therapeutic efficacy of anti-mitotic drugs.     

Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities

In C. elegans, mutants in the anaphase-promoting complex or cyclosome (APC/C) exhibit defects in germline proliferation, the formation of the vulva and male tail, and the metaphase to anaphase transition of meiosis I. Oocytes lacking APC/C activity can be fertilized but arrest in metaphase of meiosis I and are blocked from further development. To examine the cell cycle and developmental consequences of reducing but not fully depleting APC/C activity, we analyzed defects in embryos and larvae of mat-1/cdc-27 mutants grown at semi-permissive temperatures. Hypomorphic embryos developed to the multicellular stage but were slow to complete meiosis I and displayed aberrant meiotic chromosome separation. More severely affected embryos skipped meiosis II altogether and exhibited striking defects in meiotic exit. These latter embryos failed to produce normal eggshells or establish normal asymmetries prior to the first mitotic division. In developing larvae, extended M-phase delays in late-dividing cell lineages were associated with defects in the morphogenesis of the male tail. This study reveals the importance of dosage-specific mutants in analyzing molecular functions of a ubiquitously functioning protein within different cell types and tissues, and striking correlations between specific abnormalities in cell cycle progression and particular developmental defects.     

Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator

During oogenesis in metazoans, the meiotic divisions must be coordinated with development of the oocyte to ensure successful fertilization and subsequent embryogenesis. The ways in which the mitotic machinery is specialized for meiosis are not fully understood. cortex, which encodes a putative female meiosis-specific anaphase-promoting complex/cyclosome (APC/C) activator, is required for proper meiosis in Drosophila. We demonstrate that CORT physically associates with core subunits of the APC/C in ovaries. APC/C^CORT targets Cyclin A for degradation prior to the metaphase I arrest, while Cyclins B and B3 are not targeted until after egg activation. We investigate the regulation of CORT and find that CORT protein is specifically expressed during the meiotic divisions in the oocyte. Polyadenylation of cort mRNA is correlated with appearance of CORT protein at oocyte maturation, while deadenylation of cort mRNA occurs in the early embryo. CORT protein is targeted for degradation by the APC/C following egg activation, and this degradation is dependent on an intact D-box in the C terminus of CORT. Our studies reveal the mechanism for developmental regulation of an APC/C activator and suggest it is one strategy for control of the female meiotic cell cycle in a multicellular organism.     

A process independent of the anaphase-promoting complex contributes to instability of the yeast S phase cyclin Clb5

Proteolytic destruction of many cyclins is induced by a multi-subunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). In the budding yeast Saccharomyces cerevisiae, the S phase cyclin Clb5 and the mitotic cyclins Clb1-4 are known as substrates of this complex. The relevance of APC/C in proteolysis of Clb5 is still under debate. Importantly, a deletion of the Clb5 destruction box has little influence on cell cycle progression. To understand Clb5 degradation in more detail, we applied in vivo pulse labeling to determine the half-life of Clb5 at different cell cycle stages and in the presence or absence of APC/C activity. Clb5 is significantly unstable, with a half-life of approximately 8-10 min, at cell cycle periods when APC/C is inactive and in mutants impaired in APC/C function. A Clb5 version lacking its cyclin destruction box is similarly unstable. The half-life of Clb5 is further decreased in a destruction box-dependent manner to 3-5 min in mitotic or G(1) cells with active APC/C. Clb5 instability is highly dependent on the function of the proteasome. We conclude that Clb5 proteolysis involves two different modes for targeting of Clb5 to the proteasome, an APC/C-dependent and an APC/C-independent mechanism. These different modes apparently have overlapping functions in restricting Clb5 levels in a normal cell cycle, but APC/C function is essential in the presence of abnormally high Clb5 levels.