The meiosis I-to-meiosis II transition in mouse oocytes requires separase activity

Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development. The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division. In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase. Their disjunction is mediated by Separase, which cleaves the cohesin. The activation of Separase requires prior degradation of its associated inhibitor, called securin. Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome. After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase. In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity. Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes. We therefore investigated if Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes.     

Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach

Kinetochores and chromatid cores of meiotic chromosomes of the grasshopper species Arcyptera fusca and Eyprepocnemis plorans were differentially silver stained to analyse the possible involvement of both structures in chromatid cohesiveness and meiotic chromosome segregation. Special attention was paid to the behaviour of these structures in the univalent sex chromosome, and in B univalents with different orientations during the first meiotic division. It was observed that while sister chromatid of univalents are associated at metaphase I, chromatid cores are individualised independently of their orientation. We think that cohesive proteins on the inner surface of sister chromatids, and not the chromatid cores, are involved in the chromatid cohesiveness that maintains associated sister chromatids of bivalents and univalents until anaphase I. At anaphase I sister chromatids of amphitelically oriented B univalents or spontaneous autosomal univalents separate but do not reach the poles because they remain connected at the centromere by a long strand which can be visualized by silver staining, that joins stretched sister kinetochores. This strand is normally observed between sister kinetochores of half-bivalents at metaphase II and early anaphase II. We suggest that certain centromere proteins that form the silver-stainable strand assure chromosome integrity until metaphase II. These cohesive centromere proteins would be released or modified during anaphase II to allow normal chromatid segregation. Failure of this process during the first meiotic division could lead to the lagging of amphitelically oriented univalents. Based on our results we propose a model of meiotic chromosome segregation. During mitosis the cohesive proteins located at the centromere and chromosome arms are released during the same cellular division. During meiosis those proteins must be sequentially inactivated, i.e. those situated on the inner surface of the chromatids must be eliminated during the first meiotic division while those located at the centromere must be released during the second meiotic division.by D.P. Bazett-Jones     

Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes.

Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.     

Slk19p is necessary to prevent separation of sister chromatids in meiosis I

BACKGROUND: A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids remain joined, moving as a unit to one pole of the spindle rather than separating as they do in mitosis. It has long been known that the sustained linkage of sister chromatids through meiotic anaphase I is accomplished by association of the chromatids at the centromere region. The localization of the cohesin Rec8p to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, but the molecular basis for the regulation of Rec8p and sister kinetochores in meiosis remains a mystery. RESULTS: We show that the SLK19 gene product from Saccharomyces cerevisiae is essential for proper chromosome segregation during meiosis I. When slk19 mutants were induced to sporulate they completed events characteristic of meiotic prophase I, but at the first meiotic division they segregated their sister chromatids to opposite poles at high frequencies. The vast majority of these cells did not perform a second meiotic division and proceeded to form dyads (asci containing two spores). Slk19p was found to localize to centromere regions of chromosomes during meiotic prophase where it remained until anaphase I. In the absence of Slk19p, Rec8p was not maintained at the centromere region through anaphase I as it is in wild-type cells. Finally, we demonstrate that Slk19p appears to function downstream of the meiosis-specific protein Spo13p in control of sister chromatid behavior during meiosis I. CONCLUSIONS: Our results suggest that Slk19p is essential at the centromere of meiotic chromosomes to prevent the premature separation of sister chromatids at meiosis I.     

Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error

The spindle assembly checkpoint (SAC) monitors attachment to microtubules and tension on chromosomes in mitosis and meiosis. It represents a surveillance mechanism that halts cells in M-phase in the presence of unattached chromosomes, associated with accumulation of checkpoint components, in particular, Mad2, at the kinetochores. A complex between the anaphase promoting factor/cylosome (APC/C), its accessory protein Cdc20 and proteins of the SAC renders APC/C inactive, usually until all chromosomes are properly assembled at the spindle equator (chromosome congression) and under tension from spindle fibres. Upon release from the SAC the APC/C can target proteins like cyclin B and securin for degradation by the proteasome. Securin degradation causes activation of separase proteolytic enzyme, and in mitosis cleavage of cohesin proteins at the centromeres and arms of sister chromatids. In meiosis I only the cohesin proteins at the sister chromatid arms are cleaved. This requires meiosis specific components and tight regulation by kinase and phosphatase activities. There is no S-phase between meiotic divisions. Second meiosis resembles mitosis. Mammalian oocytes arrest constitutively at metaphase II in presence of aligned chromosomes, which is due to the activity of the cytostatic factor (CSF). The SAC has been identified in spermatogenesis and oogenesis, but gender-differences may contribute to sex-specific differential responses to aneugens. The age-related reduction in expression of components of the SAC in mammalian oocytes may act synergistically with spindle and other cell organelles' dysfunction, and a partial loss of cohesion between sister chromatids to predispose oocytes to errors in chromosome segregation. This might affect dose-response to aneugens. In view of the tendency to have children at advanced maternal ages it appears relevant to pursue studies on consequences of ageing on the susceptibility of human oocytes to the induction of meiotic error by aneugens and establish models to assess risks to human health by environmental exposures.     

Kiss and break up—a safe passage to anaphase in mitosis and meiosis

Eukaryotic chromosomes have many challenges to overcome between DNA replication and sister chromatid segregation. If these challenges are not met, cell death or unregulated cell division (cancer) may result. During prophase, chromosomes condense, the nuclear membrane breaks down and cohesins are removed from chromosome arms. In prometaphase, initial spindle attachments are made by sister kinetochores followed by correction of erroneous attachments, centromere oscillation between spindle poles and congression towards the cell's equator. In metaphase, all chromosomes attain stable bipolar spindle attachments and align at the metaphase plate, ready for the metaphase–anaphase transition when all ties between sister chromatids are broken. This review concentrates on recent developments that have revealed the intricacies of these processes. We now know more about how the mechanisms of cohesin removal differ between prophase and the metaphase–anaphase transition, the processes for detection and correction of improper spindle-kinetochore attachments and the concept that tension between sister kinetochores is the driving factor for satisfying the spindle checkpoint. We are also beginning to gain some understanding of the mechanisms behind the co-segregation of sister chromatids at the first meiotic division. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell‐specific conditional knockout strategy, we show that Polo‐like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.     

The role of sister chromatid cohesiveness and structure in meiotic behaviour

Sister chromatid cores, kinetochores and the connecting strand between sister kinetochores were differentially silver stained to analyse the behaviour of these structures during meiosis in normal and two spontaneous desynaptic individuals of Chorthippus jucundus (Orthoptera). In these desynaptic individuals most of the chromosomes appear as univalents and orient equationally in the first meiotic division. Despite this abnormal segregation pattern, the changes in chromosome structure follow the same timing as in normal individuals and seem to be strictly phase dependent. Chromosomes in the first prometaphase have associated sister kinetochores and sister chromatid cores that lie in the chromosome midline; we propose that this promotes the initial monopolar orientation of chromosomes. However, the requirements of tension for stable attachment to the spindle force the autosomal univalents to acquire amphitelic orientation. Sister kinetochores behave in a chromosome orientation-dependent manner and, in the first metaphase, they appear to be interconnected by a strand that can be detected by silver impregnation, as seen in the second metaphase of wild-type individuals. The disappearance of the sister kinetochore-connecting strand, needed for equational chromatid segregation, however, can only take place in the second meiotic division. This connecting strand is ultimately responsible for the inability of chromosomes to segregate sister chromatids in the first anaphase. Received: 25 March 1997; in revised form: 14 July 1997 / Accepted: 22 August 1997     

The aurora kinase AIR-2 functions in the release of chromosome cohesion in Caenorhabditis elegans meiosis

Accurate chromosome segregation during cell division requires not only the establishment, but also the precise, regulated release of chromosome cohesion. Chromosome dynamics during meiosis are more complicated, because homologues separate at anaphase I whereas sister chromatids remain attached until anaphase II. How the selective release of chromosome cohesion is regulated during meiosis remains unclear. We show that the aurora-B kinase AIR-2 regulates the selective release of chromosome cohesion during Caenorhabditis elegans meiosis. AIR-2 localizes to subchromosomal regions corresponding to last points of contact between homologues in metaphase I and between sister chromatids in metaphase II. Depletion of AIR-2 by RNA interference (RNAi) prevents chromosome separation at both anaphases, with concomitant prevention of meiotic cohesin REC-8 release from meiotic chromosomes. We show that AIR-2 phosphorylates REC-8 at a major amino acid in vitro. Interestingly, depletion of two PP1 phosphatases, CeGLC-7alpha and CeGLC-7beta, abolishes the restricted localization pattern of AIR-2. In Ceglc-7alpha/beta(RNAi) embryos, AIR-2 is detected on the entire bivalent. Concurrently, chromosomal REC-8 is dramatically reduced and sister chromatids are separated precociously at anaphase I in Ceglc-7alpha/beta(RNAi) embryos. We propose that AIR-2 promotes the release of chromosome cohesion via phosphorylation of REC-8 at specific chromosomal locations and that CeGLC-7alpha/beta, directly or indirectly, antagonize AIR-2 activity.     

Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis

Immunocytological and in situ hybridization evidence supports the hypothesis that at meiosis of chiasmate organisms, chromosomal disjunction and reductional segregation of sister centromeres are integrated with synaptonemal complex functions. The Mr 125,000 synaptic protein, Syn1, present between cores of paired homologous chromosomes during pachytene of meiotic prophase, is lost from synaptonemal complexes coordinately with homolog separation at diplotene. Separation is constrained by exchanges between non-sister chromatids, the chiasmata. We show that the Mr 30,000 chromosomal core protein, Cor1, associated with sister chromatid pairs, remains an axial component of post-pachytene chromosomes until metaphase I. We demonstrate that at this time the chromatin loops are still attached to their cores. A reciprocal exchange event between two homologous non-sister chromatids is therefore immobilized by anchorage of sister chromatids to their respective cores. Cores thus contribute to the sister chromatid cohesiveness required for maintenance of chiasmata and proper chromosomal disjunction. Cor1 protein accumulates in juxtaposition to pairs of sister centromeres during metaphase I. Presumably, independent movement of sister centromeres at anaphase I is restricted by Cor1 anchorage. That reductional separation of sister centromeres is mediated by Cor1, is supported by the dissociation of Cor1 from separating sister centromeres at anaphase II and by its absence from mitotic anaphases.     

Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol-induced parthenogenones

A high frequency of parthenogenetic activation occurs when ovulated mouse oocytes are briefly exposed to a dilute solution of ethanol in vitro. Cytogenetic analyses of parthenogenones at metaphase of the first cleavage division have confirmed that parthenogenetic activation, per se, does not increase the incidence of chromosome segregation errors during the completion of the second meiotic division. Ethanol-induced activation, however, significantly increases the incidence of aneuploidy. The ultrastructural changes that occur in the morphology and organization of the second meiotic spindle apparatus in ethanol- and hyaluronidase-activated oocytes is reported here. Abnormalities in the arrangement of microtubule arrays and chromosome position were principally observed in ethanol-activated oocytes at anaphase and telophase of the second meiotic division, but were only rarely observed in hyaluronidase-activated oocytes. It is proposed that the abnormalities in spindle morphology and chromosome displacement observed in ethanol-activated oocytes represent the initial events that lead to chromosome segregation errors following exposure to this agent.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.     

DNA catenations that link sister chromatids until the onset of anaphase are maintained by a checkpoint mechanism

Treatment of Allium cepa meristematic cells in metaphase with the topoisomerase II inhibitor ICRF-193, results in bridging of the sister chromatids at anaphase. Separation of the sisters in experimentally generated acentric chromosomal fragments was also inhibited by ICRF-193, indicating that some non-centromeric catenations also persist in metaphase chromosomes. Thus, catenations must be resolved by DNA topoisomerase II at the metaphase-to-anaphase transition to allow segregation of sisters. A passive mechanism could maintain catenations holding sisters until the onset of anaphase. At this point the opposite tension exerted on sister chromatids could render the decatenation reaction physically more favorable than catenation. But this possibility was dismissed as acentric chromosome fragments were able to separate their sister chromatids at anaphase. A timing mechanism (a common trigger for two processes taking different times to be completed) could passively couple the resolution of the last remaining catenations to the moment of anaphase onset. This possibility was also discarded as cells arrested in metaphase with microtubule-destabilising drugs still displayed anaphase bridges when released in the presence of ICRF-193. It is possible that a checkpoint mechanism prevents the release of the last catenations linking sisters until the onset of anaphase. To test whether cells are competent to fully resolve catenations before anaphase onset, we generated multinucleate plant cells. In this system, the nuclei within a single multinucleate cell displayed differences in chromosome condensation at metaphase, but initiated anaphase synchronously. When multinucleates were treated with ICRF-193 at the metaphase-toanaphase transition, tangled and untangled anaphases were observed within the same cell. This can only occur if cells are competent to disentangle sister chromatids before the onset of anaphase, but are prevented from doing so by a checkpoint mechanism.     

DNA damage response during mouse oocyte maturation

Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II. Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.     

MAPK-Activated Protein Kinase 2 Is Required for Mouse Meiotic Spindle Assembly and Kinetochore-Microtubule Attachment

MAPK-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple physiological functions in mitosis. Here, we show for the first time the unique distribution pattern of MK2 in meiosis. Phospho-MK2 was localized on bipolar spindle minus ends and along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions at metaphase of first meiosis (MI stage) in mouse oocytes. At metaphase of second meiosis (MII stage), p-MK2 was localized on the bipolar spindle minus ends and at the inner centromere region of sister chromatids as dots. Knockdown or inhibition of MK2 resulted in spindle defects. Spindles were surrounded by irregular nondisjunction chromosomes, which were arranged in an amphitelic or syntelic/monotelic manner, or chromosomes detached from the spindles. Kinetochore–microtubule attachments were impaired in MK2-deficient oocytes because spindle microtubules became unstable in response to cold treatment. In addition, homologous chromosome segregation and meiosis progression were inhibited in these oocytes. Our data suggest that MK2 may be essential for functional meiotic bipolar spindle formation, chromosome segregation and proper kinetochore–microtubule attachments.     

Mnd2, an essential antagonist of the anaphase-promoting complex during meiotic prophase

Meiotic cohesin serves in sister chromatid linkage and DNA repair until its subunit Rec8 is cleaved by separase. Separase is activated when its inhibitor, securin, is polyubiquitinated by the Cdc20 regulated anaphase-promoting complex (APC(Cdc20)) and consequently degraded. Differently regulated APCs (APC(Cdh1), APC(Ama1)) have not been implicated in securin degradation at meiosis I. We show that Mnd2, a factor known to associate with APC components, prevents premature securin degradation in meiosis by APC(Ama1). mnd2Delta cells lack linear chromosome axes and exhibit precocious sister chromatid separation, but deletion of AMA1 suppresses these defects. Besides securin, Sgo1, a protein essential for protection of centromeric cohesion during anaphase I, is also destabilized in mnd2delta cells. Mnd2's disappearance prior to anaphase II may activate APC(Ama1). Human oocytes may spend many years in meiotic prophase before maturation. Inhibitors of meiotic APC variants could prevent loss of chiasmata also in these cells, thereby guarding against aberrant chromosome segregation.     

Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes

BACKGROUND: The importance of mitotic spindle checkpoint control has been well established during somatic cell divisions. The metaphase-to-anaphase transition takes place only when all sister chromatids have been properly attached to the bipolar spindle and are aligned at the metaphase plate. Failure of this checkpoint may lead to unequal separation of sister chromatids. On the contrary, the existence of such a checkpoint during the first meiotic division in mammalian oocytes when homologous chromosomes are segregated has remained controversial. RESULTS: Here, we show that mouse oocytes respond to spindle damage by a transient and reversible cell cycle arrest in metaphase I with high Maturation Promoting Factor (MPF) activity. Furthermore, the mitotic checkpoint protein Mad2 is present throughout meiotic maturation and is recruited to unattached kinetochores. Overexpression of Mad2 in meiosis I leads to a cell cycle arrest in metaphase I. Expression of a dominant-negative Mad2 protein interferes with proper spindle checkpoint arrest. CONCLUSIONS: Errors in meiosis I cause missegregation of chromosomes and can result in the generation of aneuploid embryos with severe birth defects. In human oocytes, failures in spindle checkpoint control may be responsible for the generation of trisomies (e.g., Down Syndrome) due to chromosome missegregation in meiosis I. Up to now, the mechanisms ensuring correct separation of chromosomes in meiosis I remained unknown. Our study shows for the first time that a functional Mad2-dependent spindle checkpoint exists during the first meiotic division in mammalian oocytes.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.     

A novel yeast mutant that is defective in regulation of the Anaphase-Promoting Complex by the spindle damage checkpoint

The accurate segregation of sister chromatids at the metaphase to anaphase transition in Saccharomyces cerevisiae is regulated by the activity of the anaphase-promoting complex or cyclosome (APC/C). In the event of spindle damage or monopolar spindle attachment, the spindle checkpoint is activated and inhibits APC/C activity towards the anaphase inhibitor Pds1p, resulting in a cell cycle arrest at metaphase. We have identified a novel allele of a gene for an APC/C subunit, cdc16-183 , in S. cerevisiae. cdc16-183 mutants arrest at metaphase at 37°C, and are supersensitive to the spindle-damaging agent nocodazole, which activates the spindle checkpoint, at lower temperatures. This supersensitivity to nocodazole cannot be explained by impairment of the spindle checkpoint pathway, as cells respond normally to spindle damage with a stable metaphase arrest and high levels of Pds1p. Despite showing metaphase arrest at G2/M at 37°C, cdc16-183 mutants are able to perform tested G1 functions normally at this temperature. This is the first demonstration that a mutation in a core APC/C subunit can result in a MAD2-dependent arrest at the restrictive temperature. Our results suggest that the cdc16-183 mutant may have a novel APC/C defect(s) that mimics or activates the spindle checkpoint pathway.Communicated by C. P. Hollenberg     

Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast.

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.