Cell cycle: how, when and why cells get rid of cyclin A.

Sequences outside the 'destruction box' direct the degradation of cyclin A to completion before the metaphase-anaphase transition; cyclin A that escapes timely degradation can block the metaphase-anaphase transition, impede anaphase and telophase, and impair a cell's ability to arrest in G1 of the next cell cycle.     

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.     

Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila

The metaphase-anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells. A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle. Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy. Here we show that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies. Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants - they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis. These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast.     

Characterization and expression of mammalian cyclin b3, a prepachytene meiotic cyclin

We report the identification and expression pattern of a full-length human cDNA and a partial mouse cDNA encoding cyclin B3. Cyclin B3 (CCNB3) is conserved from Caenorhabditis elegans to Homo sapiens and has an undefined meiotic function in female, but not male Drosophila melanogaster. We show that H. sapiens cyclin B3 interacts with cdk2, is localized to the nucleus, and is degraded during anaphase entry after the degradation of cyclin B1. Degradation is dependent on sequences conserved in a destruction box motif. Overexpression of nondegradable cyclin B3 blocks the mitotic cell cycle in late anaphase, and at higher doses it can interfere with progression through G(1) and entry into S phase. H. sapiens cyclin B3 mRNA and protein are detected readily in developing germ cells in the human testis and not in any other tissue. The mouse cDNA has allowed us to further localize cyclin B3 mRNA to leptotene and zygotene spermatocytes. The expression pattern of mammalian cyclin B3 suggests that it may be important for events occurring in early meiotic prophase I.     

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition.

We show that the sequence of Drosophila cyclin B has greater identity with B-type cyclins from other animal phyla than with Drosophila cyclin A, suggesting that the two cyclins have distinct roles that have been maintained in evolution. Cyclin A is not detectable in unfertilized eggs and is present at low levels prior to cellularization of the syncytial embryo. In contrast, the levels of cyclin B remain uniformly high throughout these developmental stages. In cells within cellularized embryos and the larval brain, cyclin A accumulates to peak levels in prophase and is degraded throughout the period in which chromosomes are becoming aligned on the metaphase plate. The degradation of cyclin B, on the other hand, does not occur until the metaphase-anaphase transition. In cells arrested at c-metaphase by treating with microtubule destabilizing drugs to prevent spindle formation, cyclin A has been degraded in the arrested cells, whereas cyclin B is maintained at high levels. These observations suggest that cyclin A has a role in the G2-M transition that is independent of spindle formation, and that entry into anaphase is a key requirement for the degradation of cyclin B.     

Ca2+ signal blockers can inhibit M/A transition in mammalian cells by interfering with the spindle checkpoint

A key step in mitosis is the sister-chromatid separation at the metaphase-anaphase (M/A) transition. Several earlier studies had suggested that Ca(2+) signal is involved in regulating this process in somatic cells. The detailed mechanisms, however, are not yet well understood. In this study, we used the GFP-gene fusion method and a living-cell imaging technique to examine the effects of suppressing cytosolic Ca(2+) level on the mitotic process in HeLa and PtK2 cells. We observed that application of the Ca(2+) chelator BAPTA/AM can block or severely delay the M/A transition. This blockage was caused by a failure in activating the anaphase-promoting complex (APC), since both cyclin B and securin could not be degraded under this situation. Furthermore, using YFP-labeled tubulin, we found that the mitotic spindle structure in most of the BAPTA-treated cells gradually deformed with time. Other Ca(2+) signal blockers, such as heparin, also produced a similar effect. These results suggest that one pathway for the blockage of M/A transition by suppressing cytosolic Ca(2+) level is due to its interference with the mitotic spindle checkpoint.     

Kinetochore microtubule dynamics and the metaphase-anaphase transition

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.     

A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1

In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.     

Cell cycle-regulated proteolysis of mitotic target proteins

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.     

Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis

CENP-E is a kinesin-like protein that binds to kinetochores through the early stages of mitosis, but after initiation of anaphase, it relocalizes to the overlapping microtubules in the midzone, ultimately concentration in the developing midbody. By immunoblotting of cells separated at various positions in the cell cycle using centrifugal elutriation, we show that CENP-E levels increase progressively across the cycle peaking at approximately 22,000 molecules/cell early in mitosis, followed by an abrupt (> 10 fold) loss at the end of mitosis. Pulse-labeling with [35S]methionine reveals that beyond a twofold increase in synthesis between G1 and G2, interphase accumulation results primarily from stabilization of CENP-E during S and G2. Despite localizing in the midbody during normal cell division, CENP-E loss at the end of mitosis is independent of cytokinesis, since complete blockage of division with cytochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degradation of as yet unidentified components at the metaphase/anaphase transition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of timed proteolysis.     

Transitions regulating the timing of cytokinesis in embryonic cells

Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement for furrow initiation in animal cells. In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit. Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition. Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not. Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA. Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint.     

A Highlights from MBoC Selection: PP1 promotes cyclin B destruction and the metaphase–anaphase transition by dephosphorylating CDC20

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator CDC20 (APC/C^CDC20) form the main ubiquitin E3 ligase for these two proteins. APC/C^CDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilizes cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1 phosphorylation–defective CDC20^6A mutants. These CDC20^6A cells show a normal spindle checkpoint response and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase transition by promoting APC/C^CDC20-dependent destruction of cyclin B in human cells.     

DNA damage leads to a Cyclin A-dependent delay in metaphase-anaphase transition in the Drosophila gastrula

BACKGROUND: In response to DNA damage, fission yeast, mammalian cells, and cells of the Drosophila gastrula inhibit Cdk1 to delay the entry into mitosis. In contrast, budding yeast delays metaphase-anaphase transition by stabilization of an anaphase inhibitor, Pds1p. A variation of the second response is seen in Drosophila cleavage embryos; when nuclei enter mitosis with damaged DNA, centrosomes lose gamma-tubulin, spindles lose astral microtubules, chromosomes fail to reach a metaphase configuration, and interphase resumes without an intervening anaphase. The resulting polyploid nuclei are eliminated. RESULTS: The cells of the Drosophila gastrula can also delay metaphase-anaphase transition in response to DNA damage. This delay accompanies the stabilization of Cyclin A, a known inhibitor of sister chromosome separation in Drosophila. Unlike in cleavage embryos, gamma-tubulin remains at the spindle poles, and anaphase always occurs after the delay. Cyclin A mutants fail to delay metaphase-anaphase transition after irradiation and show an increased frequency of chromosome breakage in the subsequent anaphase. CONCLUSIONS: DNA damage delays metaphase-anaphase transition in Drosophila by stabilizing Cyclin A. This delay may normally serve to preserve chromosomal integrity during segregation. To our knowledge this is the first report of a metazoan metaphase-anaphase transition being delayed in response to DNA damage. Though mitotic progression is modulated in response to DNA damage in both cleaving and gastruating embryos of Drosophila, different mechanisms operate. These differences are discussed in the context of differential cell cycle regulation in cleavage and gastrula stages.     

Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase

Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.     

Regulation of Intracellular MEK1/2 Translocation in Mouse Oocytes: Cytoplasmic Dynein/Dynactin-Mediated Poleward Transport and Cyclin B Degradation-Dependent Release from Spindle Poles

We recently reported that MEK1/2 plays an important role in microtubule organization and spindle pole tethering in mouse oocytes, but how the intracellular transport of this protein is regulated remains unknown. In the present study, we investigated the mechanisms of poleward MEK1/2 transport during the prometaphase I/metaphase I transition and MEK1/2 release from the spindle poles during the metaphase I/anaphase I transition in mouse oocytes. Firstly, we found that p-MEK1/2 was colocalized with dynactin at the spindle poles. Inhibition of the cytoplasmic dynein/dynactin complex by antibody microinjection blocked polar accumulation of p-MEK1/2 and caused obvious spindle abnormalities. Moreover, coimmunoprecipitation of p-MEK1/2 and dynein or dynactin from mouse oocyte extracts confirmed their association at metaphase I. Secondly, disruption of microtubules by nocodazole resulted in the failure of poleward p-MEK1/2 transport. Whereas, when the nocodazole-treated oocytes were recovered in fresh culture medium, the spindle reformed and p-MEK1/2 relocalized to the spindle poles. Finally, we examined the mechanism of p-MEK1/2 release from the spindle poles. In control oocytes, polar p-MEK1/2 was gradually released during metaphase I/anaphase I transition. By contrast, in the presence of nondegradable cyclin B (△90), p-MEK1/2 still remained at the spindle poles at anaphase I. Our results indicate that poleward MEK1/2 transport is a cytoplasmic dynein/dynactin-mediated and spindle microtubule-dependent intracellular movement, and that its subsequent anaphase release from spindle poles is dependent on cyclin B degradation.     

When the genome plays dice: circumvention of the spindle assembly checkpoint and near-random chromosome segregation in multipolar cancer cell mitoses

Background

Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes. Cancer cells, however, occasionally divide into three or more directions. Such multipolar mitoses have been proposed to generate genetic diversity and thereby contribute to clonal evolution. However, this notion has been little validated experimentally.

Principal Findings

Chromosome segregation and DNA content in daughter cells from multipolar mitoses were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells. The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent nullisomies in the daughter cells. Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition. The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle. Centromeric AURKB and MAD2 foci were observed frequently to remain on the centromeres of multipolar ana-telophase chromosomes, indicating that multipolar mitoses were able to circumvent the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase. Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells.

Conclusion

The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells. Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations.     

Cdc20 Is Critical for Meiosis I and Fertility of Female Mice

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.     

Nuclear and microtubular cycles in heterophasic multinuclearTriticum root-tip cells induced by caffeine

Summary Nuclear and microtubular cycles were studied in large heterophasic multinuclear cells induced in root tips ofTriticum turgidum by caffeine treatment. Multinuclear cells and cells with polyploid nuclei exhibited various configurations of multiple and complex preprophase microtubule (Mt) bands (PPBs), including helical ones. The developmental stages of PPBs in some heterophasic cells did not comply with the cell cycle stages of the associated nuclei, a fact indicating that these events are not directly controlled by the associated nuclei. The heterophasic cells exhibited asynchronous nuclei at different stages of mitosis. In cells displaying prophase and interphase nuclei, the prophase spindle was either absent or developed around both of them or developed around the prophase nuclei earlier than around the interphase ones. During prometaphase-metaphase of the advanced nuclei the lagging interphase nuclei were induced to form prematurely condensed chromosomes (PCCs) along with spindle formation around them. These observations suggest that the mitotic transition in heterophasic cells is delayed but is ultimately achieved due to the effect of the advanced nuclei, which induces a premature mitotic entry of the lagging nuclei. Although kinetochore Mt bundles were found associated with PCCs, their metaphase and anaphase spindles were abnormal resulting in abnormal or abortive anaphases. In some heterophasic cells, metaphase-anaphase transition did not take place simultaneously in different chromosome groups, signifying that the cells do not exit from the mitotic state after anaphase initiation of the advanced nuclei. Asynchronous pace of mitosis of different chromosome groups was also observed during anaphase and telophase. Implications of these observations in understanding plant cell cycle regulation are discussed.Abbreviations cdk cyclin dependent kinase - Mt microtubule - PCC prematurely condensed chromosome - PPB preprophase band     

Inefficient degradation of cyclin B1 re-activates the spindle checkpoint right after sister chromatid disjunction

Sister chromatid separation creates a sudden loss of tension on kinetochores, which could, in principle, re-activate the spindle checkpoint in anaphase. This so-called “anaphase problem” is probably avoided by timely inactivation of cyclin B1-Cdk1, which may prevent the spindle tension sensing Aurora B kinase from destabilizing kinetochore–microtubule interactions as they lose tension in anaphase. However, exactly how spindle checkpoint re-activation is prevented remains unclear.

Here, we investigated how different degrees of cyclin B1 stabilization affected the spindle checkpoint in metaphase and anaphase. Cells expressing a strongly stabilized (R42A) mutant of cyclin B1 degraded APC/C^Cdc20 substrates normally, showing that checkpoint release was not inhibited by high cyclin B1-Cdk1 activity. However, after this initial wave of APC/C^Cdc20 activity, the spindle checkpoint returned in cells with uncohesed sister chromatids. Expression of a lysine mutant of cyclin B1 that is degraded only slightly inefficiently allowed a normal metaphase-to-anaphase transition. Strikingly, however, the spindle checkpoint returned in cells that had not degraded the cyclin B1 mutant 10–15 min after anaphase onset. When cyclin B1 remained in late anaphase, cytokinesis stalled, and translocation of INCENP from separated sister chromatids to the spindle midzone was blocked. This late anaphase arrest required the activity of Aurora B and Mps1. In conclusion, our results reveal that complete removal of cyclin B1 is essential to prevent the return of the spindle checkpoint following sister chromatid disjunction. Speculatively, increasing activity of APC/C^Cdc20 in late anaphase helps to keep cyclin B1 levels low.