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Cohesin and Cdk1: An anaphase barricade
Abstract
Separation of sister chromatids at anaphase in metazoan cells requires only the cleavage of the kleisin subunit of centromeric cohesin, but efficient poleward movement of separated sisters requires the associated loss in Cdk1 activity. Activation of the anaphase-promoting complex/cyclosome ensures these events are coordinated.
... Homologous chromosomes in meiosis I and sister chromatids in meiosis II are prevented from undergoing premature disjunction by cohesin complexes that hold the chromosomes together. At anaphase onset, the protease separase cleaves cohesion [11,12], resulting in a poleward movement of separated chromosomes, a process that also requires loss of cyclin-dependent kinase 1 (CDK1) activity [13,14]. Before anaphase, separase is inhibited through binding to a chaperone protein, securin , and at anaphase onset APC/C activity not only causes degradation of cyclin B1 but also securin, thereby activating separase. ...
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.
The comprehension of chromosome movement during mitosis and meiosis is essential for understanding genetic transmission, but students often find this process difficult to grasp in a classroom setting. I propose a “double-spring model” that incorporates a physical demonstration and can be used as a teaching tool to help students understand this process, particularly the energy changes that occur during different stages of the cell cycle. My teaching experience has demonstrated that this model is very effective, and it has been favorably received by numerous students.
One of the critical events in mitosis is proper sister chromatid cohesion, which is mediated by a protein complex called cohesin. The cohesin complex is best known for its role in tethering the sister DNA molecules. This review summarizes recent progress in understanding the functions of cohesin, and of its major subunits, SMC1, SMC3, Scc1 and Scc3. It is now clear that cohesin also plays crucial roles in controlling transcription and gene expression, DNA damage repair and spindle pole formation; functions that are beyond the traditional view of cohesin as a 'molecular glue' that holds sister chromosomes together.
The metaphase-anaphase transition is orchestrated through proteolysis of numerous proteins by a ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C). A crucial aspect of this process is sister chromatid separation, which is thought to be mediated by separase, a thiol protease activated by the APC/C. Separase cleaves cohesin, a ring-shaped complex that entraps sister DNAs. It is a matter of debate whether cohesin-independent forces also contribute to sister chromatid cohesion. Using 4D live-cell imaging of Drosophila melanogaster syncytial embryos blocked in metaphase (via APC/C inhibition), we show that artificial cohesin cleavage is sufficient to trigger chromosome disjunction. This is nevertheless insufficient for correct chromosome segregation. Kinetochore-microtubule attachments are rapidly destabilized by the loss of tension caused by cohesin cleavage in the presence of high Cdk1 (cyclin-dependent kinase 1) activity, as occurs when the APC/C cannot destroy mitotic cyclins. Metaphase chromosomes undergo a bona fide anaphase when cohesin cleavage is combined with Cdk1 inhibition. We conclude that only two key events, opening of cohesin rings and downregulation of Cdk1, are sufficient to drive proper segregation of chromosomes in anaphase.
The BubR1 checkpoint protein performs multiple functions in mitosis. We have carried out a functional analysis of conserved motifs of human BubR1 (also known as BUB1B) and demonstrate that spindle assembly checkpoint (SAC) and chromosome attachment functions can be uncoupled from each other. Mutation of five proline-directed serine phosphorylation sites, identified in vivo by mass spectrometry, essentially abolishes attachment of chromosomes to the spindle but has no effect on SAC functionality. By contrast, mutation of the two conserved KEN boxes required for SAC function does not impact chromosome congression. Interestingly, the contribution of the two KEN-box motifs is not equal. Cdc20 associates with the N-terminal but not C-terminal KEN box, and mutation of the N-terminal KEN motif results in more severe acceleration of mitotic timing. Moreover, the two KEN motifs are not sufficient for maximal binding of Cdc20 and APC/C, which also requires sequences in the BubR1 C-terminus. Finally, mutation of the GLEBS motif causes loss of Bub3 interaction and mislocalization of BubR1 from the kinetochore; concomitantly, BubR1 phosphorylation as well as SAC activity and chromosome congression are impaired, indicating that the GLEBS motif is strictly required for both major functions of human BubR1.
Successful cell division requires that chromosomes attach to opposite poles of the mitotic spindle (bi-orientation). Aurora
B kinase regulates chromosome-spindle attachments by phosphorylating kinetochore substrates that bind microtubules. Centromere
tension stabilizes bi-oriented attachments, but how physical forces are translated into signaling at individual centromeres
is unknown. Using fluorescence resonance energy transfer–based biosensors to measure localized phosphorylation dynamics in
living cells, we found that phosphorylation of an Aurora B substrate at the kinetochore depended on its distance from the
kinase at the inner centromere. Furthermore, repositioning Aurora B closer to the kinetochore prevented stabilization of bi-oriented
attachments and activated the spindle checkpoint. Thus, centromere tension can be sensed by increased spatial separation of
Aurora B from kinetochore substrates, which reduces phosphorylation and stabilizes kinetochore microtubules.
The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction.
Sister chromatid cohesion, mediated by a complex called cohesin, is crucial--particularly at centromeres--for proper chromosome segregation in mitosis and meiosis. In animal mitotic cells, phosphorylation of cohesin promotes its dissociation from chromosomes, but centromeric cohesin is protected by shugoshin until kinetochores are properly captured by the spindle microtubules. However, the mechanism of shugoshin-dependent protection of cohesin is unknown. Here we find a specific subtype of serine/threonine protein phosphatase 2A (PP2A) associating with human shugoshin. PP2A colocalizes with shugoshin at centromeres and is required for centromeric protection. Purified shugoshin complex has an ability to reverse the phosphorylation of cohesin in vitro, suggesting that dephosphorylation of cohesin is the mechanism of protection at centromeres. Meiotic shugoshin of fission yeast also associates with PP2A, with both proteins collaboratively protecting Rec8-containing cohesin at centromeres. Thus, we have revealed a conserved mechanism of centromeric protection of eukaryotic chromosomes in mitosis and meiosis.
Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres.
The assembly of mitotic chromosomes is controlled by condensin complexes. In vertebrates, condensin I binds to chromatin in prometaphase, confers rigidity to chromosomes and enables the release of cohesin complexes from chromosome arms, whereas condensin II associates with chromosomes in prophase and promotes their condensation. Both complexes are essential for chromosome segregation in anaphase. Although the association of condensins with chromatin is important for the assembly and segregation of mitotic chromosomes, it is poorly understood how this process is controlled. Here we show that the mitotic kinase Aurora B regulates the association of condensin I, but not the interaction of condensin II with chromatin. Quantitative time-lapse imaging of cells expressing GFP-tagged condensin subunits revealed that Aurora B is required for efficient loading of condensin I onto chromosomes in prometaphase and for maintenance of the complex on chromosomes in later stages of mitosis. The three non-SMC subunits of condensin I are Aurora B substrates in vitro and their mitosis-specific phosphorylation depends on Aurora B in vivo. Our data indicate that Aurora B contributes to chromosome rigidity and segregation by promoting the binding of condensin I to chromatin. We have also addressed how Aurora B might mediate the dissociation of cohesin from chromosome arms.
Mammalian oocytes begin meiosis in the fetal ovary, but only complete it when fertilized in the adult reproductive tract. This review examines the cell biology of this protracted process: from entry of primordial germ cells into meiosis to conception. The defining feature of meiosis is two consecutive cell divisions (meiosis I and II) and two cell cycle arrests: at the germinal vesicle (GV), dictyate stage of prophase I and at metaphase II. These arrests are spanned by three key events, the focus of this review: (i) passage from mitosis to GV arrest during fetal life, regulated by retinoic acid; (ii) passage through meiosis I and (iii) completion of meiosis II following fertilization, both meiotic divisions being regulated by cyclin-dependent kinase (CDK1) activity. Meiosis I in human oocytes is associated with an age-related high rate of chromosomal mis-segregation, such as trisomy 21 (Down's syndrome), resulting in aneuploid conceptuses. Although aneuploidy is likely to be multifactorial, oocytes from older women may be predisposed to be becoming aneuploid as a consequence of an age-long decline in the cohesive ties holding chromosomes together. Such loss goes undetected by the oocyte during meiosis I either because its ability to respond and block division also deteriorates with age, or as a consequence of being inherently unable to respond to the types of segregation defects induced by cohesion loss.
In eukaryotes, the spindle-assembly checkpoint (SAC) is a ubiquitous safety device that ensures the fidelity of chromosome segregation in mitosis. The SAC prevents chromosome mis-segregation and aneuploidy, and its dysfunction is implicated in tumorigenesis. Recent molecular analyses have begun to shed light on the complex interaction of the checkpoint proteins with kinetochores--structures that mediate the binding of spindle microtubules to chromosomes in mitosis. These studies are finally starting to reveal the mechanisms of checkpoint activation and silencing during mitotic progression.
The cohesin complex is a major constituent of interphase and mitotic chromosomes. Apart from its role in mediating sister chromatid cohesion, it is also important for DNA double-strand-break repair and transcriptional control. The functions of cohesin are regulated by phosphorylation, acetylation, ATP hydrolysis, and site-specific proteolysis. Recent evidence suggests that cohesin acts as a novel topological device that traps chromosomal DNA within a large tripartite ring formed by its core subunits.
In interphase, chromosomes are associated with proteins and RNAs that participate in many processes, such as DNA replication, transcription, recombination and repair of DNA damage. These components (for example, cohesin) might have to be removed during mitosis, as they might become obstacles that inhibit chromosome segregation or reduce its fidelity. Such a clearing mechanism that operates along mitotic chromosomes might require proteins that are implicated in chromosome segregation. I propose that condensin and DNA topoisomerase II (TOP2), as well as separase, help to clear the way for mitosis.
The survival of eukaryotes depends on the accurate coordination of mitosis with cytokinesis. Key for the coordination of both processes is the chromosomal passenger complex (CPC) comprising Aurora-B, INCENP, survivin, and borealin. The translocation of the CPC from centromeres to the spindle midzone, a structure composed of antiparallel microtubules, at anaphase onset is critical for the completion of cytokinesis. In mammalian cells, the mitotic kinesin Mklp2 is essential for recruitment of the CPC to the spindle midzone. However, the mechanism regulating the binding of Mklp2 to microtubules has remained unknown. Here, we demonstrate that Mklp2 and the CPC mutually depend on each other for midzone localization; i.e., Mklp2 is mislocalized in INCENP-RNAi cells and vice versa. Remarkably, INCENP is required for localization of Mklp2 to the ends of stable microtubules in cells with low Cdk1 activity. In vitro assays revealed that the association between the CPC and Mklp2 is negatively regulated by Cdk1. Collectively, our data suggest that anaphase onset triggers the association between the CPC and Mklp2 and that this association targets the CPC-Mklp2 complex to the ends of stable microtubules in the spindle midzone.
In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This "sister chromatid cohesion" is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on the multisubunit cohesin complex, which possibly forms the physical bridges connecting sisters. Proteolytic cleavage of cohesin's Sccl subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin. We show here that separin is a cysteine protease related to caspases that alone can cleave Sccl in vitro. Cleavage of Sccl in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles.
The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. We show that Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis.
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.
Cohesin establishes sister-chromatid cohesion from S phase until mitosis or meiosis. To allow chromosome segregation, cohesion has to be dissolved. In vertebrate cells, this process is mediated in part by the protease separase, which destroys a small amount of cohesin, but most cohesin is removed from chromosomes without proteolysis. How this is achieved is poorly understood. Here, we show that the interaction between cohesin and chromatin is controlled by Wapl, a protein implicated in heterochromatin formation and tumorigenesis. Wapl is associated with cohesin throughout the cell cycle, and its depletion blocks cohesin dissociation from chromosomes during the early stages of mitosis and prevents the resolution of sister chromatids until anaphase, which occurs after a delay. Wapl depletion also increases the residence time of cohesin on chromatin in interphase. Our data indicate that Wapl is required to unlock cohesin from a particular state in which it is stably bound to chromatin.