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Sister chromatid cohesion is mediated by the cohesin complex. In mitotic prophase cohesin is removed from chromosome arms in a Wapl- and phosphorylation-dependent manner. Sgo1-PP2A protects pericentromeric cohesion by dephosphorylation of cohesin and its associated Wapl antagonist sororin. However, Sgo1-PP2A relocates to inner kinetochores well before sister chromatids are separated by separase, leaving pericentromeric regions unprotected. Why deprotected cohesin is not removed by Wapl remains enigmatic. By reconstituting Wapl-dependent cohesin removal from chromatin in vitro, we discovered a requirement for Nek2a and Cdk1/2-cyclin A2. These kinases phosphorylate cohesin-bound Pds5b, thereby converting it from a sororin- to a Wapl-interactor. Replacement of endogenous Pds5b by a phosphorylation mimetic variant causes premature sister chromatid separation (PCS). Conversely, phosphorylation-resistant Pds5b impairs chromosome arm separation in prometaphase-arrested cells and suppresses PCS in the absence of Sgo1. Early mitotic degradation of Nek2a and cyclin A2 may therefore explain why only separase, but not Wapl, can trigger anaphase., (© 2024. The Author(s).)

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.

Prolonged mitosis often results in apoptosis 1 . Shortened mitosis causes tumorigenic aneuploidy, but it is unclear whether it also activates the apoptotic machinery 2 . Separase, a cysteine protease and trigger of all eukaryotic anaphases, has a caspase-like catalytic domain but has not previously been associated with cell death 3,4 . Here we show that human cells that enter mitosis with already active separase rapidly undergo death in mitosis owing to direct cleavage of anti-apoptotic MCL1 and BCL-XL by separase. Cleavage not only prevents MCL1 and BCL-XL from sequestering pro-apoptotic BAK, but also converts them into active promoters of death in mitosis. Our data strongly suggest that the deadliest cleavage fragment, the C-terminal half of MCL1, forms BAK/BAX-like pores in the mitochondrial outer membrane. MCL1 and BCL-XL are turned into separase substrates only upon phosphorylation by NEK2A. Early mitotic degradation of this kinase is therefore crucial for preventing apoptosis upon scheduled activation of separase in metaphase. Speeding up mitosis by abrogation of the spindle assembly checkpoint results in a temporal overlap of the enzymatic activities of NEK2A and separase and consequently in cell death. We propose that NEK2A and separase jointly check on spindle assembly checkpoint integrity and eliminate cells that are prone to chromosome missegregation owing to accelerated progression through early mitosis.

Separation of eukaryotic sister chromatids during the cell cycle is timed by the spindle assembly checkpoint (SAC) and ultimately triggered when separase cleaves cohesion-mediating cohesin 1-3 . Silencing of the SAC during metaphase activates the ubiquitin ligase APC/C (anaphase-promoting complex, also known as the cyclosome) and results in the proteasomal destruction of the separase inhibitor securin 1 . In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions 4,5 . Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma 6,7 , is turned into a separase inhibitor upon association with SAC-activated MAD2. SGO2-MAD2 can functionally replace securin and sequesters most separase in securin-knockout cells. Acute loss of securin and SGO2, but not of either protein individually, resulted in separase deregulation associated with premature cohesin cleavage and cytotoxicity. Similar to securin 8,9 , SGO2 is a competitive inhibitor that uses a pseudo-substrate sequence to block the active site of separase. APC/C-dependent ubiquitylation and action of the AAA-ATPase TRIP13 in conjunction with the MAD2-specific adaptor p31 comet liberate separase from SGO2-MAD2 in vitro. The latter mechanism facilitates a considerable degree of sister chromatid separation in securin-knockout cells that lack APC/C activity. Thus, our results identify an unexpected function of SGO2 in mitotically dividing cells and a mechanism of separase regulation that is independent of securin but still supervised by the SAC.

Separase, a cysteine protease of the CD clan, triggers chromosome segregation during mitosis by cleaving the cohesin ring entrapping the two sister chromatids. Deregulated separase activity is associated with aneuploidy, a hallmark of most human cancers. In fact, separase is highly overexpressed in many solid cancers, making it an attractive chemotherapeutic target. To identify small molecules capable of inhibiting separase in its complex cellular environment, we established a highly sensitive assay to quantify separase activity in cells and screened a 51 009-member library for separase inhibitors. In vitro assays confirmed that the identified compounds efficiently inhibited separase, while not affecting caspase-1, another CD-clan protease structurally related to separase. Importantly, HeLa cells with compromised separase activity displayed severe chromosome segregation defects upon compound treatment, confirming that the identified inhibitors are bioactive in tumor tissue culture cells. Structure-activity relationship studies succeeded in the optimization of the most promising inhibitor. Overall, this study demonstrates the feasibility of identifying separase-specific inhibitors, which serve as promising lead compounds for the development of clinically relevant separase inhibiting drugs.

The multisubunit ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for mitosis by promoting timely degradation of cyclin B1. APC/C is tightly regulated by the spindle assembly checkpoint (SAC), which involves MPS1 and MAD2-dependent temporal inhibition of APC/C. We analyzed the contribution of the APC/C subunits APC7 and APC16 to APC/C composition and function in human cells. APC16 is required for APC7 assembly into APC/C, whereas APC16 assembles independently of APC7. APC7 and APC16 knockout cells display no major defects in mitotic progression, cyclin B1 degradation, or SAC response, but APC/C lacking these two subunits shows reduced ubiquitylation activity in vitro. Strikingly, deletion of APC7 or APC16 is sufficient to provide synthetic viability to MAD2 deletion. ΔAPC7ΔMAD2 cells display accelerated mitosis and require SAC-independent MPS1 function for genome stability. These findings reveal that the composition of APC/C critically influences the importance of the SAC in humans., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double-strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology-directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation., (© 2018 The Authors.)

The DNA-embracing, ring-shaped multiprotein complex cohesin mediates sister chromatid cohesion and is stepwise displaced in mitosis by Wapl and separase (also known as ESPL1) to facilitate anaphase. Proper regulation of chromosome cohesion throughout meiosis is critical for preventing formation of aneuploid gametes, which are associated with trisomies and infertility in humans. Studying cohesion in meiocytes is complicated by their difficult experimental amenability and the absence of cohesin turnover. Here, we use cultured somatic cells to unravel fundamental aspects of meiotic cohesin. When expressed in Hek293 cells, the kleisin Rec8 displays no affinity for the peripheral cohesin subunits Stag1 or Stag2 and remains cytoplasmic. However, co-expression of Stag3 is sufficient for Rec8 to enter the nucleus, load onto chromatin, and functionally replace its mitotic counterpart Scc1 (also known as RAD21) during sister chromatid cohesion and dissolution. Rec8-Stag3 cohesin physically interacts with Pds5, Wapl and sororin (also known as CDCA5). Importantly, Rec8-Stag3 cohesin is shown to be susceptible to Wapl-dependent ring opening and sororin-mediated protection. These findings exemplify that our model system is suitable to rapidly generate testable predictions for important unresolved issues of meiotic cohesion regulation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

Organelles in eukaryotic cells often have complex shapes that deviate significantly from simple spheres. A prime example is the endoplasmic reticulum (ER) that forms an extensive network of membrane tubules in many mammalian cell types and in reconstitution assays in vitro. Despite the successful hunt for molecular determinants of ER shape we are still far from having a comprehensive understanding of ER network morphogenesis. Here, we have studied the hitherto neglected influence of the host substrate when reconstituting ER networks in vitro as compared to ER networks in vivo. In culture cells we observed cytoplasm-spanning ER networks with tubules being connected almost exclusively by three-way junctions and segment lengths being narrowly distributed around a mean length of about 1μm. In contrast, networks reconstituted from purified ER microsomes on flat glass or gel substrates of varying stiffness showed significantly broader length distributions with an up to fourfold larger mean length. Self-assembly of ER microsomes on small oil droplets, however, yielded networks that resembled more closely the native ER network of mammalian cells. We conclude from these observations that the ER microsomes' inherent self-assembly capacity is sufficient to support network formation with a native geometry if the influence of the host substrate's surface chemistry becomes negligible. We hypothesize that under these conditions the networks' preference for three-way junctions follows from creating 'starfish-shaped' vesicles when ER microsomes with a protein-induced spontaneous curvature undergo fusion., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Vertebrate immature oocytes are arrested at prophase of meiosis I (MI). Hormonal stimulation breaks this prophase-I arrest and induces re-entry into MI. The mechanism underlying meiotic resumption remains largely elusive. Here, we demonstrate that the anaphase-promoting complex/cyclosome (APC/C) in complex with Cdh1 has an unexpected function in meiosis in that it is essential for meiotic resumption. We identify the catalytic subunit of protein phosphatase 6 (PP6c) as the critical substrate whose APC/C(Cdh1)-mediated destruction is a prerequisite for the re-entry of immature Xenopus laevis oocytes into MI. Preventing PP6c destruction impairs activating autophosphorylation of Aurora A, a cell-cycle kinase critical for meiotic translation. Restoring meiotic translation rescues the meiotic resumption defect of Cdh1-depleted oocytes. Thus, our studies discover that the essential function of the APC/C in triggering cell-cycle transitions is not limited to M-phase exit but also applies to entry into meiotic M-phase, and identify a crucial APC/C-PP6c-Aurora A axis in the resumption of female meiosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Separation of human sister chromatids involves the removal of DNA embracing cohesin ring complexes. Ring opening occurs by prophase-pathway-dependent phosphorylation and separase-mediated cleavage, with the former being antagonized at centromeres by Sgo1-dependent PP2A recruitment. Intriguingly, prophase pathway signaling and separase's proteolytic activity also bring about centriole disengagement, whereas Sgo1 is again counteracting this licensing step of later centrosome duplication. Here, we demonstrate that alternative splice variants of human Sgo1 specifically and exclusively localize and function either at centromeres or centrosomes. A small C-terminal peptide encoded by exon 9 of SGO1 (CTS for centrosomal targeting signal of human Sgo1) is necessary and sufficient to drive centrosomal localization and simultaneously abrogate centromeric association of corresponding Sgo1 isoforms. Cohesin is shown to be a target of the prophase pathway at centrosomes and protected by Sgo1-PP2A. Accordingly, premature centriole disengagement in response to Sgo1 depletion is suppressed by blocking ring opening of an engineered cohesin., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Ring-shaped cohesin keeps sister chromatids paired until cleavage of its Scc1/Rad21 subunit by separase triggers chromosome segregation in anaphase. Vertebrate separase is held inactive by mutually exclusive binding to securin or Cdk1-cyclin B1 and becomes unleashed only upon ubiquitin-dependent degradation of these regulators. Although most separase is usually found in association with securin, this anaphase inhibitor is dispensable for murine life while Cdk1-cyclin B1-dependent control of separase is essential. Here, we show that securin-independent inhibition of separase by Cdk1-cyclin B1 in early mitosis requires the phosphorylation-specific peptidyl-prolyl cis/trans isomerase Pin1. Furthermore, isomerization of previously securin-bound separase at the metaphase-to-anaphase transition renders it resistant to re-inhibition by residual securin. At the same time, isomerization also limits the half-life of separase's proteolytic activity, explaining how cohesin can be reloaded onto telophase chromatin in the absence of securin and cyclin B1 without being cleaved., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sister chromatid cohesion is established during replication by entrapment of both dsDNAs within the cohesin ring complex. It is dissolved in anaphase when separase, a giant cysteine endopeptidase, cleaves the Scc1/Rad21 subunit of cohesin, thereby triggering chromosome segregation. Separase is held inactive by association with securin until this anaphase inhibitor is destroyed at the metaphase-to-anaphase transition by ubiquitin-dependent degradation. The relevant ubiquitin ligase, the anaphase-promoting complex/cyclosome, also targets cyclin B1, thereby causing inactivation of Cdk1 and mitotic exit. Although separase is essential, securin knock-out mice are surprisingly viable and fertile. Capitalizing on our previous finding that Cdk1-cyclin B1 can also bind and inhibit separase, we investigated whether this kinase might be suitable to maintain faithful timing and execution of anaphase in the absence of securin. We found that, similar to securin, Cdk1-cyclin B1 regulates separase in both a positive and negative manner. Although securin associates with nascent separase to co-translationally assist proper folding, Cdk1-cyclin B1 acts on native state separase. Upon entry into mitosis, Cdk1-cyclin B1-dependent phosphorylation of Ser-1126 renders separase prone to inactivation by aggregation/precipitation. Stable association of Cdk1-cyclin B1 with phosphorylated separase counteracts this tendency and stabilizes separase in an inhibited yet activatable state. These opposing effects are suited to prevent premature cleavage of cohesin in early mitosis while ensuring timely activation of separase by anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. Coupling sister chromatid separation with subsequent exit from mitosis by this simplified mode might have been the common scheme of mitotic control prior to the evolution of securin., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase-associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase-associated securin is destabilized by introduction of phosphorylation-mimetic aspartates or extinction of separase-associated PP2A activity. G2- or prometaphase-arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate-mutant securin. Thus, PP2A-dependent stabilization of separase-associated securin prevents precocious activation of separase during checkpoint-mediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase., (© 2014 The Authors.)

Accurate chromosome segregation depends on coordination between cohesion resolution and kinetochore-microtubule interactions (K-fibers), a process regulated by the spindle assembly checkpoint (SAC). How these diverse processes are coordinated remains unclear. We show that in mammalian oocytes Shugoshin-like protein 2 (Sgol2) in addition to protecting cohesin, plays an important role in turning off the SAC, in promoting the congression and bi-orientation of bivalents on meiosis I spindles, in facilitating formation of K-fibers and in limiting bivalent stretching. Sgol2's ability to protect cohesin depends on its interaction with PP2A, as is its ability to silence the SAC, with the latter being mediated by direct binding to Mad2. In contrast, its effect on bivalent stretching and K-fiber formation is independent of PP2A and mediated by recruitment of MCAK and inhibition of Aurora C kinase activity respectively. By virtue of its multiple interactions, Sgol2 links many of the processes essential for faithful chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.01133.001.

Faithful transmission of chromosomes during eukaryotic cell division requires sister chromatids to be paired from their generation in S phase until their separation in M phase. Cohesion is mediated by the cohesin complex, whose Smc1, Smc3 and Scc1 subunits form a tripartite ring that entraps both DNA double strands. Whereas centromeric cohesin is removed in late metaphase by Scc1 cleavage, metazoan cohesin at chromosome arms is displaced already in prophase by proteolysis-independent signalling. Which of the three gates is triggered by the prophase pathway to open has remained enigmatic. Here, we show that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates. In contrast, loading of human cohesin onto chromatin in telophase occurs through the Smc1-Smc3 hinge. We propose that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesin ring.

In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication, CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors, including CDC-45 and GINS. During the elongation step, the CDC-45/GINS complex moves with the replication fork; however, it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48(UFD-1/NPL-4)-deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

Mammalian centromeric cohesin is protected from phosphorylation-dependent displacement in mitotic prophase by shugoshin-1 (Sgo1), while shugoshin-2 (Sgo2) protects cohesin from separase-dependent cleavage in meiosis I. In higher eukaryotes, progression and faithful execution of both mitosis and meiosis are controlled by the spindle assembly checkpoint, which delays anaphase onset until chromosomes have achieved proper attachment to microtubules. According to the so-called template model, Mad1-Mad2 complexes at unattached kinetochores instruct conformational change of soluble Mad2, thus catalysing Mad2 binding to its target Cdc20. Here, we show that human Sgo2, but not Sgo1, specifically interacts with Mad2 in a manner that strongly resembles the interactions of Mad2 with Mad1 or Cdc20. Sgo2 contains a Mad1/Cdc20-like Mad2-interaction motif and competes with Mad1 and Cdc20 for binding to Mad2. NMR and biochemical analyses show that shugoshin binding induces similar conformational changes in Mad2 as do Mad1 or Cdc20. Mad2 binding regulates fine-tuning of Sgo2's sub-centromeric localization. Mad2 binding is conserved in the only known Xenopus laevis shugoshin homologue and, compatible with a putative meiotic function, the interaction occurs in oocytes.

Mature Xenopus oocytes are arrested in meiosis by the activity of XErp1/Emi2, an inhibitor of the ubiquitin-ligase anaphase-promoting complex/cyclosome (APC/C). On fertilization, XErp1 is degraded, resulting in APC/C activation and the consequent degradation of cell-cycle regulators and exit from meiosis. In this study, we show that a modest increase in the activity of the ubiquitin-conjugating enzyme UbcX overrides the meiotic arrest in an APC/C-dependent reaction. Intriguingly, XErp1 remains stable in these conditions. We found that UbcX causes the ubiquitylation of XErp1, followed by its dissociation from the APC/C. Our data support the idea that ubiquitylation regulates the APC/C-inhibitory activity of XErp1.

Sister chromatid cohesion is mediated by DNA catenation and proteinaceous cohesin complexes. The recent visualization of PICH (Plk1-interacting checkpoint helicase)-coated DNA threads in anaphase cells raises new questions as to the role of DNA catenation and its regulation in time and space. In the present study we show that persistent DNA catenation induced by inhibition of Topoisomerase-IIalpha can contribute to sister chromatid cohesion in the absence of cohesin complexes and that resolution of catenation is essential for abscission. Furthermore, we use an in vitro chromatid separation assay to investigate the temporal and functional relationship between cohesin removal and Topoisomerase-IIalpha-mediated decatenation. Our data suggest that centromere decatenation can occur only after separase activation and cohesin removal, providing a plausible explanation for the persistence of centromere threads after anaphase onset.

Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific alpha-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females.

Mammalian eggs remain arrested at metaphase of the second meiotic division (metII) for an indeterminate time before fertilization. During this period, which can last several hours, the continued attachment of sister chromatids is thought to be achieved by inhibition of the protease separase. Separase is known to be inhibited by binding either securin or Maturation (M-Phase)-Promoting Factor, a heterodimer of CDK1/cyclin B1. However, the relative contribution of securin and CDK/cyclin B1 to sister chromatid attachment during metII arrest has not been assessed. Although there are conditions in which either CDK1/cyclinB1 activity or securin can prevent sister chromatid disjunction, principally by overexpression of non-degradable cyclin B1 or securin, we find here that separase activity is primarily regulated by securin and not CDK1/cyclin B1. Thus the CDK1 inhibitor roscovitine and an antibody we designed to block the interaction of CDK1/cyclin B1 with separase, both failed to induce sister disjunction. In contrast, securin morpholino knockdown specifically induced loss of sister attachment, that could be restored by securin cRNA rescue. During metII arrest separase appears primarily regulated by securin binding, not CDK1/cyclin B1.

NMR-based drug screening methods provide the most reliable characterization of binding propensities of ligands to their target proteins. They are, however, one of the least effective methods in terms of the amount of protein required and the time needed for acquiring an NMR experiment. We show here that the introduction of tryptophan to proteins permits rapid screening by monitoring a simple 1D proton NMR signal of the NH side chain ((N)H(epsilon)) of the tryptophan. The method could also provide quantitative characterization of the antagonist-protein and antagonist-protein-protein interactions in the form of KDs and fractions of the released proteins from their mutual binding. We illustrate the method with the lead compounds that block the Mdm2-p53 interaction and by studying inhibitors that bind to cyclin-dependent kinase 2 (CDK2).

Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.

Sister chromatids are held together by the ring-shaped cohesin complex, which likely entraps both DNA-double strands in its middle. This tie is resolved in anaphase when separase, a giant protease, becomes active and cleaves the kleisin subunit of cohesin. Premature activation of separase and, hence, chromosome missegregation are prevented by at least two inhibitory mechanisms. Although securin has long been appreciated as a direct inhibitor of separase, surprisingly its loss has basically no phenotype in mammals. Phosphorylation-dependent binding of Cdk1 constitutes an alternative way to inhibit vertebrate separase. Its importance is illustrated by the premature loss of cohesion when Cdk1-resistant separase is expressed in mammalian cells without or with limiting amounts of securin. Here, we demonstrate that crucial inhibitory phosphorylations occur within a region of human separase that is also shown to make direct contact with the cyclin B1 subunit of Cdk1. This region exhibits a weak homology to Saccharomyces cerevisiae Cdc6 of similar Cdk1 binding behavior, thereby establishing phosphoserine/threonine-mediated binding of partners as a conserved characteristic of B-type cyclins. In contrast to the Cdc6-like domain, the previously identified serine 1126 phosphorylation is fully dispensable for Cdk1 binding to separase fragments. This suggests that despite its in vivo relevance, it promotes complex formation indirectly, possibly by inducing a conformational change in full-length separase.

The onset of anaphase is triggered by the activation of a site-specific protease called separase. Separase cleaves the chromosomal cohesins holding the duplicated sister chromatids together, allowing sisters to simultaneously separate and segregate to opposite ends of the cell before division. Activated separase cleaves not only cohesin, but also itself; however, the biological significance of separase self-cleavage has remained elusive. Before anaphase, separase is inhibited by at least two mechanisms. The first involves the binding of securin, whereas the second requires the phosphorylation-dependent binding of cyclin-dependent kinase 1 (Cdk1)/cyclin B1. Because securin and Cdk1/cyclin B1 interact with separase in a mutually exclusive manner, the degradation of both these inhibitors plays an important role in activating separase at anaphase. Here we identify a new separase interacting partner, a specific subtype of the heterotrimeric protein phosphatase 2A (PP2A). PP2A associates with separase through the B' (B56) regulatory subunit and does so independently of securin and cyclin B1 binding. The association of PP2A with separase requires a 55-amino acid domain closely juxtaposed to separase autocleavage sites. Strikingly, mutation of these cleavage sites increases PP2A binding, suggesting that separase cleavage disrupts the interaction of PP2A with separase. Furthermore, expression of a non-cleavable separase, but not a non-cleavable mutant that cannot bind PP2A, causes a premature loss of centromeric cohesion. Together these observations provide a new mechanistic insight into a physiological function for separase self-cleavage.

The highly abundant AAA-ATPase p97 is required for diverse cellular processes, of which ER-associated protein degradation (ERAD) is understood best. Previously, a new role of p97 in spindle disassembly at the end of mitosis has been reported. However, we show that neither addition of dominant-negative p97 mutants nor depletion of crucial p97 adaptors impairs transition of meiotic spindles into interphase arrays of microtubules. The dominant-negative approach is validated by inhibition of ERAD, which we reconstitute for the first time in the powerful biochemical system of Xenopus egg extracts. The role of p97 in spindle disassembly during meiotic exit should therefore be reconsidered.

Separase not only triggers anaphase of meiosis I by proteolytic cleavage of cohesin on chromosome arms, but in vitro vertebrate separase also acts as a direct inhibitor of cyclin-dependent kinase 1 (Cdk1) on liberation from the inhibitory protein, securin. Blocking separase-Cdk1 complex formation by microinjection of anti-separase antibodies prevents polar-body extrusion in vertebrate oocytes. Importantly, proper meiotic maturation is rescued by chemical inhibition of Cdk1 or expression of Cdk1-binding separase fragments lacking cohesin-cleaving activity.

Chromosome segregation in mitosis and meiosis is triggered by activation of a large protease, separase. While it has been known for some time that the anaphase inhibitor securin regulates separase activity recent work shows that this is only half the story. In vertebrates Cdk1-dependent inhibition of separase represents a second, securin-independent branch of anaphase regulation. Furthermore, an unanticipated ability of separase to inhibit Cdk1 suggests additional, nonproteolytic functions of separase.

Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin(-/-) cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

Stable maintenance of genetic information requires chromosome segregation to occur with high accuracy. Anaphase is triggered when ring-shaped cohesin is cleaved by separase, a protease regulated by association with its inhibitor securin. Dispensability of vertebrate securin strongly suggests additional means of separase regulation. Indeed, sister chromatid separation but not securin degradation is inhibited by constitutively active cyclin-dependent kinase 1 (Cdk1) and can be rescued solely by preventing phosphorylation of separase. We demonstrate that Cdk1-dependent phosphorylation of separase is not sufficient for inhibition. In a second step, Cdk1 stably binds phosphorylated separase via its regulatory cyclin B1 subunit. Complex formation results in inhibition of both protease and kinase, and we show that vertebrate separase is a direct inhibitor of Cdk1. This unanticipated function of separase is negatively regulated by securin but independent of separase's proteolytic activity.

After the degradation of its inhibitor securin, separase initiates chromosome segregation during the metaphase-to-anaphase transition by cleaving cohesin. Here we present a density map at a resolution of 25 A of negatively stained separase-securin complex. Based on labeling data and sequence analysis, we propose a model for the structure of separase, consisting of 26 ARM repeats, an unstructured region of 280 residues and two caspase-like domains, with securin binding to the ARM repeats.

Cleavage of the ring-like cohesin complex by separase triggers segregation of sister chromatids in anaphase. This simplistic model has recently been extended by exciting discoveries on three levels: regulation of anaphase by posttranslational modifications and the cohesin protector shugoshin; non-proteolytic roles of separase; and cohesin-independent linkage of sister chromatids.

Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast. Here, we investigate how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex. As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively. Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro. Furthermore, deletion of corresponding phosphorylation sites results in viable cells. Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes. Instead, the formation of active Ctf13p/Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion. skp1 mutants exhibit growth defects on media containing geldanamycin. A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality. An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p.

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is approximately 9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent approximately 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

This experiment provides a test for the common sense knowledge that moderate physical exercise leads to mood improvement. Furthermore, it was tested whether light exercise intensifies negative feeling states or alleviates them. 30 female and 30 male students of psychology served as subjects (mean age 25.3 years, SD = 4.8). After being exposed to a mood induction procedure designed to elicit either a positive or negative feeling state, the subjects had to pedal a bicycle ergometer with 0, 50 or 75 Watt load. Cardiovascular variables and self-reports of mood states were assessed during a baseline period, after the mood induction, following the ergometer exercise, and after a follow-up period. The mood induction procedures were successful, but only for a short duration. Physiological activation was observed according to the ergometer loadings. Moderate physical exercise led to an increase of positive feeling states (Concentration) and a decrease of negative feeling states (Tiredness) in the follow-up period. Corresponding changes in tension related states could not be observed, probably due to the weakness of the mood induction procedure employed and the low level of energetic activation reached.