Destruction Of Securin Research Articles

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

A prometaphase mechanism of securin destruction is essential for meiotic progression in mouse oocytes

Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes.

First meiotic anaphase requires Cep55-dependent inhibitory cyclin-dependent kinase 1 phosphorylation.

During mitosis, anaphase is triggered by anaphase-promoting complex (APC)-mediated destruction of securin and cyclin B1, which leads to inactivation of cyclin-dependent kinase 1 (Cdk1). By regulating APC activity, the mitotic spindle assembly checkpoint (SAC) therefore has robust control over anaphase timing to prevent chromosome mis-segregation. Mammalian oocytes are prone to aneuploidy, the reasons for which remain obscure. In mitosis, Cep55 is required post-anaphase for the final steps of cytokinesis. We found that Cep55-depleted mouse oocytes progress normally through early meiosis I, but that anaphase I fails as a result of persistent Cdk1 activity. Unexpectedly, Cdk1 inactivation was compromised following Cep55 depletion, despite on-time SAC silencing and intact APC-mediated proteolysis. We found that impaired Cdk1 inactivation was caused by inadequate inhibitory Cdk1 phosphorylation consequent upon failure to suppress Cdc25 phosphatase, identifying a proteolysis-independent step necessary for anaphase I. Thus, the SAC in oocytes does not exert exclusive control over anaphase I initiation, providing new insight into vulnerability to error.

Generation of a Spindle Checkpoint Arrest from Synthetic Signaling Assemblies

SummaryThe spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules and ensuring high-fidelity chromosome segregation [1, 2, 3]. The checkpoint is activated by unattached kinetochores, and Mps1 kinase phosphorylates KNL1 on conserved MELT motifs to generate a binding site for the Bub3-Bub1 complex [4, 5, 6, 7]. This leads to dynamic kinetochore recruitment of Mad proteins [8, 9], a conformational change in Mad2 [10, 11, 12], and formation of the mitotic checkpoint complex (MCC: Cdc20-Mad3-Mad2 [13, 14, 15]). MCC formation inhibits the anaphase-promoting complex/cyclosome (Cdc20-APC/C), thereby preventing the proteolytic destruction of securin and cyclin and delaying anaphase onset. What happens at kinetochores after Mps1-dependent Bub3-Bub1 recruitment remains mechanistically unclear, and it is not known whether kinetochore proteins other than KNL1 have significant roles to play in checkpoint signaling and MCC generation. Here, we take a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1Spc7 and Mps1Mph1 is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest. We demonstrate that a Mad1-Bub1 complex is formed during synthetic checkpoint signaling. Analysis of bub3Δ mutants demonstrates that Bub3 acts to suppress premature checkpoint signaling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analyzing several mutants that affect localization of checkpoint complexes, we conclude that spindle checkpoint arrest can be independent of their kinetochore, spindle pole, and nuclear envelope localization.

MASTL promotes cyclin B1 destruction by enforcing Cdc20-independent binding of cyclin B1 to the APC/C.

When cells enter mitosis, the anaphase-promoting complex/cyclosome (APC/C) is activated by phosphorylation and binding of Cdc20. The RXXL destruction box (D-box) of cyclin B1 only binds Cdc20 after release of the spindle checkpoint in metaphase, initiating cyclin B1 ubiquitination upon chromosome bi-orientation. However, we found that cyclin B1, through Cdk1 and Cks, is targeted to the phosphorylated APC/CCdc20 at the start of prometaphase, when the spindle checkpoint is still active. Here, we show that MASTL is essential for cyclin B1 recruitment to the mitotic APC/C and that this occurs entirely independently of Cdc20. Importantly, MASTL-directed binding of cyclin B1 to spindle checkpoint-inhibited APC/CCdc20 critically supports efficient cyclin B1 destruction after checkpoint release. A high incidence of anaphase bridges observed in response to MASTL RNAi may result from cyclin B1 remaining after securin destruction, which is insufficient to keep MASTL-depleted cells in mitosis but delays the activation of separase.

Sharpening the anaphase switch.

The segregation of sister chromatids during mitosis is one of the most easily visualized, yet most remarkable, events during the life cycle of a cell. The accuracy of this process is essential to maintain ploidy during cell duplication. Over the past 20years, substantial progress has been made in identifying components of both the kinetochore and the mitotic spindle that generate the force to move mitotic chromosomes. Additionally, we now have a reasonable, albeit incomplete, understanding of the molecular and biochemical events that are involved in establishing and dissolving sister-chromatid cohesion. However, it is less well-understood how this dissolution of cohesion occurs synchronously on all chromosomes at the onset of anaphase. At the centre of the action is the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that, in association with its activator cell-division cycle protein 20 homologue (Cdc20), is responsible for the destruction of securin. This leads to the activation of separase, a specialized protease that cleaves the kleisin-subunit of the cohesin complex, to relieve cohesion between sister chromatids. APC/C-Cdc20 is also responsible for the destruction of cyclin B and therefore inactivation of the cyclin B-cyclin-dependent kinase 1 (Cdk1). This latter event induces a change in the microtubule dynamics that results in the movement of sister chromatids to spindle poles (anaphase A), spindle elongation (anaphase B) and the onset of cytokinesis. In the present paper, we review the emerging evidence that multiple, spatially and temporally regulated feedback loops ensure anaphase onset is rapid, co-ordinated and irreversible.

Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila.

Chromosome segregation in meiosis is controlled by a conserved pathway that culminates in Separase-mediated cleavage of the α-kleisin Rec8, leading to dissolution of cohesin rings. Drosophila has no gene encoding Rec8, and the absence of a known Separase target raises the question of whether Separase and its regulator Securin (Pim in Drosophila) are important in Drosophila meiosis. Here, we investigate the role of Securin, Separase and the cohesin complex in female meiosis using fluorescence in situ hybridization against centromeric and arm-specific sequences to monitor cohesion. We show that Securin destruction and Separase activity are required for timely release of arm cohesion in anaphase I and centromere-proximal cohesion in anaphase II. They are also required for release of arm cohesion on polar body chromosomes. Cohesion on polar body chromosomes depends on the cohesin components SMC3 and the mitotic α-kleisin Rad21 (also called Vtd in Drosophila). We provide cytological evidence that SMC3 is required for arm cohesion in female meiosis, whereas Rad21, in agreement with recent findings, is not. We conclude that in Drosophila meiosis, cohesion is regulated by a conserved Securin-Separase pathway that targets a diverged Separase target, possibly within the cohesin complex.

Activation of the APC/C Ubiquitin Ligase by Enhanced E2 Efficiency

The anaphase-promoting complex/cyclosome (APC/C) is a protein-ubiquitin ligase (E3) that initiates the final events of mitosis by catalyzing the ubiquitination and proteasomal destruction of securin, cyclins, and other substrates [1, 2]. Like other members of the RING family of E3s [3, 4], the APC/C catalyzes direct ubiquitin transfer from an E2-ubiquitin conjugate (E2-Ub) to lysine residues on the protein substrate. The APC/C is activated at specific cell-cycle stages by association with an activator subunit, Cdc20 or Cdh1, which provides binding sites for specific substrate sequence motifs, or degrons. Activator might also stimulate catalytic activity [5, 6], but the underlying mechanisms are not known. Here, we dissected activator function using an artificial fusion substrate in which the N-terminal region of securin was linked to an APC/C core subunit. This fusion substrate bound tightly to the APC/C and was ubiquitinated at a low rate in the absence of activator. Ubiquitination of this substrate was stimulated by activator, due primarily to a dramatic stimulation of E2 sensitivity (Km) and catalytic rate (kcat), which together resulted in a 670-fold stimulation of kcat/Km. Thus, activator is not simply a substrate adaptor, but also enhances catalysis by promoting a more efficient interaction with the E2-Ub. Interestingly, full E2 stimulation required activator interaction with degron motifs on the substrate. We conclude that formation of a complete APC/C-activator-substrate complex leads to a major enhancement of E2 efficiency, providing an unusual substrate-assisted catalytic mechanism that limits efficient ubiquitin transfer to specific substrates.

Cdk1 Inactivation Terminates Mitotic Checkpoint Surveillance and Stabilizes Kinetochore Attachments in Anaphase

SummaryTwo mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/CCdc20) [2]. Upon satisfaction of both pathways, the APC/CCdc20 elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/CCdc20 is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/CCdc20 couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit.

P31comet inactivates the chemically induced Mad2-dependent spindle assembly checkpoint and leads to resistance to anti-mitotic drugs

Mad2 is a key component of the spindle assembly checkpoint (SAC) that delays the onset of anaphase until all kinetochores are attached to the spindle. It binds to Cdc20 and prevents it from promoting destruction of an anaphase inhibitor, Securin. Previously, we showed that a Mad2-binding protein, p31comet, formed a complex with Mad2 upon the completion of spindle attachment. Here, we showed that the overexpression of p31comet can abolish the Mad2-dependent SAC that is induced by anti-mitotic drugs, including nocodazole, taxol, and monastrol; these drugs, except monastrol, cause aneuploidy in HeLa cells. In the absence of Eg5, which is a target of monastrol, overexpression of p31comet caused premature destruction of Securin and premature sister chromatid separation, but it did not cause aneuploidy. These results indicated that Eg5 kinesin function might be required for checkpoint exit and mitotic progression. Moreover, overexpression of p31comet led to resistance against apoptosis that was induced by nocodazole and taxol in human cells, and taxol resistance was dependent on the p31comet/Mad2 protein expression level ratio of in cancer cell lines. These results indicated that p31comet is an indicator of resistance to anti-mitotic drugs in cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-562) contains supplementary material, which is available to authorized users.

Mechanism of CRL4Cdt2, a PCNA-dependent E3 ubiquitin ligase

Eukaryotic cell cycle transitions are driven by E3 ubiquitin ligases that catalyze the ubiquitylation and destruction of specific protein targets. For example, the anaphase-promoting complex/cyclosome (APC/C) promotes the exit from mitosis via destruction of securin and mitotic cyclins, whereas CRL1(Skp2) allows entry into S phase by targeting the destruction of the cyclin-dependent kinase (CDK) inhibitor p27. Recently, an E3 ubiquitin ligase called CRL4(Cdt2) has been characterized, which couples proteolysis to DNA synthesis via an unusual mechanism that involves display of substrate degrons on the DNA polymerase processivity factor PCNA. Through its destruction of Cdt1, p21, and Set8, CRL4(Cdt2) has emerged as a master regulator that prevents rereplication in S phase. In addition, it also targets other factors such as E2F and DNA polymerase η. In this review, we discuss our current understanding of the molecular mechanism of substrate recognition by CRL4(Cdt2) and how this E3 ligase helps to maintain genome integrity.

Conserved CDC20 Cell Cycle Functions Are Carried out by Two of the Five Isoforms in Arabidopsis thaliana

BackgroundThe CDC20 and Cdh1/CCS52 proteins are substrate determinants and activators of the Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase and as such they control the mitotic cell cycle by targeting the degradation of various cell cycle regulators. In yeasts and animals the main CDC20 function is the destruction of securin and mitotic cyclins. Plants have multiple CDC20 gene copies whose functions have not been explored yet. In Arabidopsis thaliana there are five CDC20 isoforms and here we aimed at defining their contribution to cell cycle regulation, substrate selectivity and plant development.Methodology/Principal FindingsStudying the gene structure and phylogeny of plant CDC20s, the expression of the five AtCDC20 gene copies and their interactions with the APC/C subunit APC10, the CCS52 proteins, components of the mitotic checkpoint complex (MCC) and mitotic cyclin substrates, conserved CDC20 functions could be assigned for AtCDC20.1 and AtCDC20.2. The other three intron-less genes were silent and specific for Arabidopsis. We show that AtCDC20.1 and AtCDC20.2 are components of the MCC and interact with mitotic cyclins with unexpected specificity. AtCDC20.1 and AtCDC20.2 are expressed in meristems, organ primordia and AtCDC20.1 also in pollen grains and developing seeds. Knocking down both genes simultaneously by RNAi resulted in severe delay in plant development and male sterility. In these lines, the meristem size was reduced while the cell size and ploidy levels were unaffected indicating that the lower cell number and likely slowdown of the cell cycle are the cause of reduced plant growth.Conclusions/SignificanceThe intron-containing CDC20 gene copies provide conserved and redundant functions for cell cycle progression in plants and are required for meristem maintenance, plant growth and male gametophyte formation. The Arabidopsis-specific intron-less genes are possibly “retrogenes” and have hitherto undefined functions or are pseudogenes.

The FEAR network

Mitosis is governed by the oscillation of cyclin dependent kinase (CDK) activity and ubiquitin-dependent proteolysis. Entry into mitosis is initiated by mitotic cyclin-CDK activation. Anaphase onset occurs upon activation of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that promotes the destruction of the anaphase inhibitor Securin. Destruction of Securin initiates chromosome segregation by activation of the protease Separase, allowing it to cleave a subunit of the cohesin complexes that hold the duplicated sister chromatids together. Upon completion of nuclear division cells exit from mitosis, a process defined by the inactivation of CDKs, disassembly of the mitotic spindle, and cytokinesis. In the budding yeast S. cerevisiae, a signaling network known as the FEAR network is critical to ensure accurate anaphase chromosome segregation and the integration of this process with other anaphase events. Here, we summarize what is known about the regulation and function of the FEAR network in budding yeast and discuss the potential for conserved FEAR network functions in other eukaryotes.

Polo Kinase and Separase Regulate the Mitotic Licensing of Centriole Duplication in Human Cells

It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. Here we test whether such a mechanism exists in intact human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. We identified Polo-like kinase 1 (Plk1) as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. Our results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes.

Recruitment of separase to mitotic chromosomes is regulated by Aurora B

Accurate segregation of chromosomes, initiated by abrupt and irreversible dissolution of sister-chromatid cohesion at anaphase, is crucial for the faithful inheritance of parental genomes during eukaryotic cell division. The dissolution of sister-chromatid cohesion is catalyzed by separase after the destruction of securin by the anaphase-promoting complex. However, separase was localized to the mitotic centrosome, raising the question how separase hydrolyzes sister-chromatid cohesion at the centromere at anaphase onset. Here we show that separase is associated with mitotic chromosomes and this association is regulated by Aurora B kinase. Using a panel of separase antibodies, we found that separase protein accumulated in mitosis and was degraded at the end of telophase. To study the spatiotemporal distribution of separase in mitosis, we carried out immunofluorescence microscopic analyses. Surprisingly, separase was found to be associated with mitotic chromosomes from prophase to metaphase and dissociated from the chromosomes in anaphase right after sister chromatid separation. Staining of isolated mitotic chromosomes from Nocodazole-arrested cells revealed that separase is concentrated at the centromere. To examine if any mitotic kinases are responsible for chromosomal localization of separase in mitosis, we carried out RNAi-mediated knockdown and found that association of separase with mitotic chromosomes was a function of Aurora B. Consistent with the phenotype seen in the Aurora B-repressed cells, inhibition of Aurora B kinase by hersperadin prevented the association of separase with chromosomes. Our results suggest that Aurora B kinase activity helps coordinate the association of separase with chromosomes and the initiation of sister-chromatid separation.

Lin9 Is Required for Mitosis and Cell Survival during Early Zebrafish Development

LIN9 has been described as a regulator of G(1)/S and G(2)/M progression of the cell cycle in invertebrates and human cell lines. To elucidate the in vivo function of LIN9 during vertebrate development, we took advantage of the teleost zebrafish (Danio rerio). By means of antisense morpholinos we show here that Lin9-depleted embryonic cells accumulate in mitosis. Flow cytometry and confocal microscopy data demonstrate that the delay in mitotic progression is followed by apoptosis, which strongly manifests in the developing central nervous system. In accordance with these findings, we identified a cohort of Lin9-regulated genes required for different mitotic processes, including mitotic entry, metaphase/anaphase transition, and cytokinesis. Our data establish LIN9 as an essential regulator of mitosis in vertebrate development.

Regulation of APC/C Activity in Oocytes by a Bub1-Dependent Spindle Assembly Checkpoint

Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes. Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks. Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1(F) allele. Loss of Bub1 greatly accelerates resolution of chiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres. By using a quantitative assay for APC/C-mediated securin destruction, we show that the APC/C is activated in an exponential fashion, with activity peaking 12-13 hr after GVBD, and that this process is advanced by 5 hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/C's Apc2 subunit or separase. Finally, we show that Bub1's kinase domain is not required to delay APC/C activation. We conclude that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr.

Cyclin-Specific Control of Ribosomal DNA Segregation

Following chromosome duplication in S phase of the cell cycle, the sister chromatids are linked by cohesin. At the onset of anaphase, separase cleaves cohesin and thereby initiates sister chromatid separation. Separase activation results from the destruction of its inhibitor, securin, which is triggered by a ubiquitin ligase called the anaphase-promoting complex (APC). Here, we show in budding yeast that securin destruction and, thus, separase activation are not sufficient for the efficient segregation of the repetitive ribosomal DNA (rDNA). We find that rDNA segregation also requires the APC-mediated destruction of the S-phase cyclin Clb5, an activator of the protein kinase Cdk1. Mutations that prevent Clb5 destruction are lethal and cause defects in rDNA segregation and DNA synthesis. These defects are distinct from the mitotic-exit defects caused by stabilization of the mitotic cyclin Clb2, emphasizing the importance of cyclin specificity in the regulation of late-mitotic events. Efficient rDNA segregation, both in mitosis and meiosis, also requires APC-dependent destruction of Dbf4, an activator of the protein kinase Cdc7. We speculate that the dephosphorylation of Clb5-specific Cdk1 substrates and Dbf4-Cdc7 substrates drives the resolution of rDNA in early anaphase. The coincident destruction of securin, Clb5, and Dbf4 coordinates bulk chromosome segregation with segregation of rDNA.

The Spindle Checkpoint Functions of Mad3 and Mad2 Depend on a Mad3 KEN Box-mediated Interaction with Cdc20-Anaphase-promoting Complex (APC/C)

Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.

More Feedback Keeps It Going

The orderly and unidirectional progress through the four phases of the eukaryotic cell cycle that allows one cell to become two is governed by multiple feedback loops (see Santos and Ferrell). Two positive feedback loops--one in the G1 phase (Skotheim et al .) and one in anaphase (Holt et al .)--have been revealed by single-cell analysis. Both groups studied the budding yeast Saccharomyces cerevisiae , in which cyclin-dependent kinases (CDKs) and their associated regulatory proteins, the cyclins (Cln), are at the heart of the cell cycle. Skotheim et al . monitored an unstable green fluorescent protein (GFP) under the control of the CLN2 promoter to determine the rate at which the CLN2 promoter was turned on in wild-type cells and in cells lacking CLN2 and CLN1 ( cln1Δcln2Δ ). In the previous models based on studies of synchronized populations of cells, regulation of Cdk1 by only the G1 cyclin Cln3 appeared to be required for progression through the G1 Start checkpoint, which initiates transcription of the G1/S regulon that includes the CLN1 and CLN2 genes. However, Skotheim et al . found that the time from the "birth" of a daughter cell to the activation of the CLN2 promoter was much shorter and less variable in wild-type cells than in the cln1Δcln2Δ cells and that a higher proportion of cln1Δcln2Δ cells failed to bud. Translocation from the nucleus of the transcriptional inhibitor Whi5, which contributes to activation of the G1/S regulon and is a substrate of Cdk1, was delayed in cln1Δcln2Δ cells; this delay may contribute to the delays and variability observed for the expression of other genes of the G1/S regulon. Thus, although not essential for cell cycle entry, Cln1 and Cln2 appear to serve as part of a positive feedback loop that dictates the timing of progression through G1. Holt et al . investigated events near the end of the cell cycle, specifically the separation of sister chromatids during anaphase. Chromatids are held together by a protein called securin, which is targeted for degradation by the ubiquitin ligase APC (anaphase-promoting complex), which, once degraded, allows the protease separase to cleave cohesin, thereby separating the chromatids. Separase also activates the phosphatase Cdc14, which removes phosphates from Cdk1 substrates. Holt et al . identified a new Cdk1 phosphorylation site in the N-terminal domain of securin near the destruction box that is important for APC recognition (other Cdk1 phosphorylation sites were already known in the C-terminal domain). When securin was phosphorylated by Cdk1 in vitro, it was a poor substrate for APC, and dephosphorylation by Cdc14 restored the ubiquitination of securin by APC. By tagging two chromosomes with GFP-repressor fusion proteins at specific loci, the authors quantified chromosome segregation in single cells. In wild-type cells, there was a consistent 90-second delay between separation of chromosomes IV and V. In cells in which Cdc14 activity was reduced, Cdk1 activity was increased, or a securin that could not be phosphorylated by Cdk1 was present, the separation of the two chromosomes was delayed. Thus, securin, separase, and APC appear to function in a positive feedback loop that stimulates securin's degradation by activating the phosphatase Cdc14, which promotes securin's destruction. This cycle appears to contribute to the synchronicity of chromosome segregation during anaphase. J. M. Skotheim, S. Di Talia, E. D. Siggia, F. R. Cross, Positive feedback of G1 cyclins ensures coherent cell cycle entry. Nature 454 , 291-296 (2008). [PubMed] L. J. Holt, A. N. Krutchinsky, D. O. Morgan, Positive feedback sharpens the anaphase switch. Nature 454 , 353-357 (2008). [PubMed] S. D. M. Santos, J. E. Ferrell, On the cell cycle and its switches. Nature 454 , 288-289 (2008). [PubMed]