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• W2043983559 abstract "•Checkpoint proteins enrich at kinetochores quickly, but APC/C inhibition is slow•SAC signaling in anaphase is too slow to inhibit degradation of APC/C substrates•The checkpoint remains activatable even after the APC/C becomes active•Slow SAC signaling may protect anaphase while cyclin B levels are still high Chromosome attachment to the mitotic spindle in early mitosis is guarded by an Aurora B kinase-dependent error correction mechanism [1Lampson M.A. Cheeseman I.M. Sensing centromere tension: Aurora B and the regulation of kinetochore function.Trends Cell Biol. 2011; 21: 133-140Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 2Carmena M. Wheelock M. Funabiki H. Earnshaw W.C. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis.Nat. Rev. Mol. Cell Biol. 2012; 13: 789-803Crossref PubMed Scopus (563) Google Scholar] and by the spindle assembly checkpoint (SAC), which delays cell-cycle progression in response to errors in chromosome attachment [3Lara-Gonzalez P. Westhorpe F.G. Taylor S.S. The spindle assembly checkpoint.Curr. Biol. 2012; 22: R966-R980Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 4Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1721) Google Scholar]. The abrupt loss of sister chromatid cohesion at anaphase creates a type of chromosome attachment that in early mitosis would be recognized as erroneous, would elicit Aurora B-dependent destabilization of kinetochore-microtubule attachment, and would activate the checkpoint [5Oliveira R.A. Nasmyth K. Getting through anaphase: splitting the sisters and beyond.Biochem. Soc. Trans. 2010; 38: 1639-1644Crossref PubMed Scopus (29) Google Scholar, 6Vázquez-Novelle M.D. Mirchenko L. Uhlmann F. Petronczki M. The ‘anaphase problem’: how to disable the mitotic checkpoint when sisters split.Biochem. Soc. Trans. 2010; 38: 1660-1666Crossref PubMed Scopus (15) Google Scholar]. However, in anaphase, none of these responses occurs, which is vital to ensure progression through anaphase and faithful chromosome segregation. The difference has been attributed to the drop in CDK1/cyclin B activity that accompanies anaphase and causes Aurora B translocation away from centromeres [7Parry D.H. Hickson G.R. O’Farrell P.H. Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase.Curr. Biol. 2003; 13: 647-653Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 8Pereira G. Schiebel E. Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14.Science. 2003; 302: 2120-2124Crossref PubMed Scopus (220) Google Scholar, 9Hümmer S. Mayer T.U. Cdk1 negatively regulates midzone localization of the mitotic kinesin Mklp2 and the chromosomal passenger complex.Curr. Biol. 2009; 19: 607-612Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10Mirchenko L. Uhlmann F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Vázquez-Novelle M.D. Petronczki M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase.Curr. Biol. 2010; 20: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Oliveira R.A. Hamilton R.S. Pauli A. Davis I. Nasmyth K. Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei.Nat. Cell Biol. 2010; 12: 185-192Crossref PubMed Scopus (127) Google Scholar] and to the inactivation of the checkpoint by the time of anaphase [10Mirchenko L. Uhlmann F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Vázquez-Novelle M.D. Petronczki M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase.Curr. Biol. 2010; 20: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Tinker-Kulberg R.L. Morgan D.O. Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage.Genes Dev. 1999; 13: 1936-1949Crossref PubMed Scopus (147) Google Scholar, 14Palframan W.J. Meehl J.B. Jaspersen S.L. Winey M. Murray A.W. Anaphase inactivation of the spindle checkpoint.Science. 2006; 313: 680-684Crossref PubMed Scopus (101) Google Scholar]. Here, we show that checkpoint inactivation may not be crucial because checkpoint activation by anaphase chromosomes is too slow to take effect on the timescale during which anaphase is executed. In addition, we observe that checkpoint activation can still occur for a considerable time after the anaphase-promoting complex/cyclosome (APC/C) becomes active, raising the question whether the checkpoint is indeed completely inactivated by the time of anaphase under physiologic conditions. Chromosome attachment to the mitotic spindle in early mitosis is guarded by an Aurora B kinase-dependent error correction mechanism [1Lampson M.A. Cheeseman I.M. Sensing centromere tension: Aurora B and the regulation of kinetochore function.Trends Cell Biol. 2011; 21: 133-140Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 2Carmena M. Wheelock M. Funabiki H. Earnshaw W.C. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis.Nat. Rev. Mol. Cell Biol. 2012; 13: 789-803Crossref PubMed Scopus (563) Google Scholar] and by the spindle assembly checkpoint (SAC), which delays cell-cycle progression in response to errors in chromosome attachment [3Lara-Gonzalez P. Westhorpe F.G. Taylor S.S. The spindle assembly checkpoint.Curr. Biol. 2012; 22: R966-R980Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 4Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1721) Google Scholar]. The abrupt loss of sister chromatid cohesion at anaphase creates a type of chromosome attachment that in early mitosis would be recognized as erroneous, would elicit Aurora B-dependent destabilization of kinetochore-microtubule attachment, and would activate the checkpoint [5Oliveira R.A. Nasmyth K. Getting through anaphase: splitting the sisters and beyond.Biochem. Soc. Trans. 2010; 38: 1639-1644Crossref PubMed Scopus (29) Google Scholar, 6Vázquez-Novelle M.D. Mirchenko L. Uhlmann F. Petronczki M. The ‘anaphase problem’: how to disable the mitotic checkpoint when sisters split.Biochem. Soc. Trans. 2010; 38: 1660-1666Crossref PubMed Scopus (15) Google Scholar]. However, in anaphase, none of these responses occurs, which is vital to ensure progression through anaphase and faithful chromosome segregation. The difference has been attributed to the drop in CDK1/cyclin B activity that accompanies anaphase and causes Aurora B translocation away from centromeres [7Parry D.H. Hickson G.R. O’Farrell P.H. Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase.Curr. Biol. 2003; 13: 647-653Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 8Pereira G. Schiebel E. Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14.Science. 2003; 302: 2120-2124Crossref PubMed Scopus (220) Google Scholar, 9Hümmer S. Mayer T.U. Cdk1 negatively regulates midzone localization of the mitotic kinesin Mklp2 and the chromosomal passenger complex.Curr. Biol. 2009; 19: 607-612Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10Mirchenko L. Uhlmann F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Vázquez-Novelle M.D. Petronczki M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase.Curr. Biol. 2010; 20: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Oliveira R.A. Hamilton R.S. Pauli A. Davis I. Nasmyth K. Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei.Nat. Cell Biol. 2010; 12: 185-192Crossref PubMed Scopus (127) Google Scholar] and to the inactivation of the checkpoint by the time of anaphase [10Mirchenko L. Uhlmann F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Vázquez-Novelle M.D. Petronczki M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase.Curr. Biol. 2010; 20: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Tinker-Kulberg R.L. Morgan D.O. Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage.Genes Dev. 1999; 13: 1936-1949Crossref PubMed Scopus (147) Google Scholar, 14Palframan W.J. Meehl J.B. Jaspersen S.L. Winey M. Murray A.W. Anaphase inactivation of the spindle checkpoint.Science. 2006; 313: 680-684Crossref PubMed Scopus (101) Google Scholar]. Here, we show that checkpoint inactivation may not be crucial because checkpoint activation by anaphase chromosomes is too slow to take effect on the timescale during which anaphase is executed. In addition, we observe that checkpoint activation can still occur for a considerable time after the anaphase-promoting complex/cyclosome (APC/C) becomes active, raising the question whether the checkpoint is indeed completely inactivated by the time of anaphase under physiologic conditions. In several organisms, artificial maintenance of high cyclin B levels in anaphase results in recruitment of spindle assembly checkpoint proteins to kinetochores when sister chromatids split [7Parry D.H. Hickson G.R. O’Farrell P.H. Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase.Curr. Biol. 2003; 13: 647-653Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Mirchenko L. Uhlmann F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Oliveira R.A. Hamilton R.S. Pauli A. Davis I. Nasmyth K. Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei.Nat. Cell Biol. 2010; 12: 185-192Crossref PubMed Scopus (127) Google Scholar]. Yet, whether this kinetochore recruitment indeed creates a signal sufficient to inhibit the anaphase-promoting complex/cyclosome (APC/C) has remained unclear, and the observation that APC/C substrates are degraded in this situation suggests that it may not [15Wolf F. Wandke C. Isenberg N. Geley S. Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells.EMBO J. 2006; 25: 2802-2813Crossref PubMed Scopus (89) Google Scholar]. To systematically test this, we expressed physiologic amounts of nondegradable cyclin B (ΔN-Cdc13) in fission yeast (S. pombe) mitosis (Figure S1A available online). As expected [16Yamano H. Gannon J. Hunt T. The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.EMBO J. 1996; 15: 5268-5279Crossref PubMed Scopus (136) Google Scholar], sister chromatids separated in anaphase but cells maintained short metaphase-like spindles (pseudometaphase; Figure S1B). The Aurora B kinase (S.p. Ark1) was retained on centromeres (Figure 1A), and the Polo kinase Plo1 was retained on spindle pole bodies (Figures S1B and S1C), indicating that CDK1 activity remained high [17Dischinger S. Krapp A. Xie L. Paulson J.R. Simanis V. Chemical genetic analysis of the regulatory role of Cdc2p in the S. pombe septation initiation network.J. Cell Sci. 2008; 121: 843-853Crossref PubMed Scopus (58) Google Scholar, 18Mulvihill D.P. Petersen J. Ohkura H. Glover D.M. Hagan I.M. Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe.Mol. Biol. Cell. 1999; 10: 2771-2785Crossref PubMed Scopus (123) Google Scholar]. As had been observed in other cell types [7Parry D.H. Hickson G.R. O’Farrell P.H. Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase.Curr. Biol. 2003; 13: 647-653Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Oliveira R.A. Hamilton R.S. Pauli A. Davis I. Nasmyth K. Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei.Nat. Cell Biol. 2010; 12: 185-192Crossref PubMed Scopus (127) Google Scholar, 15Wolf F. Wandke C. Isenberg N. Geley S. Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells.EMBO J. 2006; 25: 2802-2813Crossref PubMed Scopus (89) Google Scholar], kinetochore attachment became unstable, and centromeres frequently detached from the spindle poles (Figures S1B and S1D). Consistent with the destabilization of kinetochore attachment, the checkpoint proteins Mad1, Mad2, Mad3, and Bub3 localized to kinetochores of pseudometaphase cells (Figures 1A and 1B). Rebinding of Mad3 was almost immediate with anaphase onset and occurred even before detachment of kinetochores from the spindle poles was observed (Figures 1B, 1C, S1D, and S1F). However, the accumulation of checkpoint proteins did not appear to inhibit the APC/C, since degradation of the APC/C substrates cyclin B (S.p. Cdc13; Figure 1D) and securin (Figures 1D and 1E) continued unhindered. This suggested that the checkpoint does not detectably block APC/C activity when cyclin B is maintained, although checkpoint proteins enrich at kinetochores. To determine at which step checkpoint signaling may be blocked, we analyzed the formation of the mitotic checkpoint complex (MCC). The MCC is the ultimate inhibitor formed by the spindle assembly checkpoint [3Lara-Gonzalez P. Westhorpe F.G. Taylor S.S. The spindle assembly checkpoint.Curr. Biol. 2012; 22: R966-R980Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar] and consists of the APC/C activator Cdc20 (S.p. Slp1) and the checkpoint proteins Mad2 and Mad3. We synchronized cells expressing nondegradable cyclin B at the G2/M transition and immunoprecipitated the APC/C subunit Lid1 (Apc4) from cells in metaphase (20 min after release) or in pseudometaphase (28 min after release) (Figures 2A, S2A, and S2B). In pseudometaphase, but not in metaphase, Mad2 was clearly associated with APC/C and Slp1, indicative of MCC formation. In contrast, in the absence of nondegradable cyclin B, Mad2 did not accumulate on the APC/C during anaphase. The amount of MCC formed in pseudometaphase seemed substantial, as we coimmunoprecipitated less Slp1 and Mad2 from a culture where the checkpoint was engaged by treatment with the microtubule-destabilizing drug MBC (Figures S2C and S2D). Hence, MCC formation does take place when cyclin B levels remain high, but seems unable to prevent degradation of APC/C substrates. Separase overexpression overrides a mitotic checkpoint arrest in budding yeast [10Mirchenko L. Uhlmann F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13Tinker-Kulberg R.L. Morgan D.O. Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage.Genes Dev. 1999; 13: 1936-1949Crossref PubMed Scopus (147) Google Scholar]. Because anaphase coincides with separase activation, we reasoned that separase might block a late step in checkpoint activity. To test this hypothesis, we conditionally overexpressed separase. In this situation, separase is not reliably inhibited by securin, and sister chromatids split almost instantaneously when cells enter mitosis (Figure 2B). Cohesin mutations that induce a similar precocious loss of cohesion cause a checkpoint-dependent delay in mitosis [19Biggins S. Murray A.W. The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint.Genes Dev. 2001; 15: 3118-3129Crossref PubMed Scopus (327) Google Scholar, 20Windecker H. Langegger M. Heinrich S. Hauf S. Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin.EMBO Rep. 2009; 10: 1022-1028Crossref PubMed Scopus (34) Google Scholar]. If separase activity was sufficient to block checkpoint signaling, separase-mediated cohesion loss should not delay cells in mitosis [10Mirchenko L. Uhlmann F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar]. However, we observed a mitotic delay in separase-overexpressing cells, which furthermore was checkpoint dependent as deletion of the checkpoint gene mad2+ abolished the delay (Figures 2B and 2C). Hence, we conclude that separase activity does not inactivate the spindle assembly checkpoint in fission yeast. The MCC formation in cells undergoing anaphase in the presence of nondegradable cyclin B (Figure 2A) suggested that checkpoint reactivation is not completely blocked, but that either the APC/C had become refractory to inhibition or that checkpoint reactivation is too slow to manifest in a block of securin degradation, i.e., the block in APC/C activity would only occur at a time when securin is completely degraded. To test the latter idea, we gave cells more time between the onset of anaphase and the completion of securin degradation. We overexpressed securin to about eight times its wild-type level (Figure S3A). This increased the total time of securin degradation from 5.5 min to 8.2 min (Figures 1E and 3A ). However, the time between anaphase and complete securin degradation was still similar to wild-type cells (∼3.7 min; Figures S3B and S3D). We therefore shifted the time of anaphase by co-overexpressing separase. As a result, anaphase occurred soon after the start of securin degradation and the time between the onset of anaphase and complete securin degradation was prolonged (7.6 min on average; Figures S3B and S3D). Interestingly, if we additionally stabilized cyclin B, securin degradation was halted before complete degradation (Figures 3A–3C). This depended on checkpoint activity, since deletion of the checkpoint gene mad2+ allowed securin degradation to run to completion (Figures 3A–3C). Hence, the checkpoint indeed reactivates at anaphase if cyclin B is stabilized, but is slow in inhibiting the APC/C: it took between 3.5 min and 16 min (average 6.5 min) from anaphase to the stabilization of securin (Figure 3D). In cells solely expressing nondegradable cyclin B, which we had monitored earlier (Figure 1E), the time from anaphase to completion of securin degradation was only 1.5 min to 4.8 min (Figure S3D). Hence, securin degradation was completed or almost completed by the time the APC/C became inhibited, which explains our failure to observe securin stabilization in this background. Since the time from anaphase onset to checkpoint protein recruitment was short (Figures 1B and 1C), this indicated that the time from checkpoint protein recruitment to APC/C inhibition is long. To test this directly, we monitored Mad2-mCherry signals in securin- and separase-overexpressing cells and related their kinetochore reoccurrence to the stabilization of securin (Figures 3E and 3F). As we had seen before, Mad2 enriched at kinetochores very soon after anaphase (0 to 60 s with an average of 30 s; see Figure 3E for an example), whereas it took 2.7 to 6 min (average 4.3 min) from the first enrichment of Mad2 until securin degradation was halted (Figure 3F). Hence, if anaphase occurs in the presence of nondegradable cyclin B, checkpoint protein rerecruitment to kinetochores is fast but APC/C inhibition is slow. Our results suggested that—although stabilization of cyclin B levels allows reactivation of the checkpoint in anaphase—reactivation is too slow to take effect before securin is degraded. It had previously been proposed that the checkpoint is completely inactivated by the time of anaphase [6Vázquez-Novelle M.D. Mirchenko L. Uhlmann F. Petronczki M. The ‘anaphase problem’: how to disable the mitotic checkpoint when sisters split.Biochem. Soc. Trans. 2010; 38: 1660-1666Crossref PubMed Scopus (15) Google Scholar, 14Palframan W.J. Meehl J.B. Jaspersen S.L. Winey M. Murray A.W. Anaphase inactivation of the spindle checkpoint.Science. 2006; 313: 680-684Crossref PubMed Scopus (101) Google Scholar]. We therefore asked whether checkpoint activation became impossible or at least more inefficient when we allowed cyclin B levels to drop. To study this systematically, we turned to cells containing the kinesin-5 mutation cut7-446 [21Hagan I. Yanagida M. Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene.Nature. 1990; 347: 563-566Crossref PubMed Scopus (274) Google Scholar] and slightly impaired kinesin function by incubating at semipermissive temperature. We reasoned that this would cause spindle instability and frequent destabilization of chromosome attachments. Indeed, in some cells, we observed securin stabilization after the start of securin degradation (Figure 4). This stabilization appeared to be related to checkpoint signaling, because in the minutes preceding stabilization, strong Mad2 signals were observed, whereas in the minutes preceding securin degradation (either initially or when restarting), Mad2 signals were low (Figure 4C). This corroborates observations by the Pines and Gerlich groups, who showed that the checkpoint can still be activated after the APC/C has become active [22Dick A.E. Gerlich D.W. Kinetic framework of spindle assembly checkpoint signalling.Nat. Cell Biol. 2013; 15: 1370-1377Crossref PubMed Scopus (122) Google Scholar, 23Hagting A. Den Elzen N. Vodermaier H.C. Waizenegger I.C. Peters J.-M. Pines J. Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.J. Cell Biol. 2002; 157: 1125-1137Crossref PubMed Scopus (263) Google Scholar]. Interestingly, the time between observing Mad2 signals and stabilization of securin levels was between 3 and 7 min (average 5.2 min), which is similar to the timing that we observed in cells with stabilized cyclin B (between 2.7 and 6 min; Figure 3F). When we plotted these values relative to the time that had elapsed since securin degradation started (Figure 4D), there was no obvious prolongation in the time needed for APC/C inhibition. This suggests that cyclin B degradation does not drastically alter the kinetics of checkpoint signaling, at least for about 2 min after the APC/C has become active. Because it takes around 2 min from the onset of securin degradation to anaphase in wild-type cells (Figure 1E), this raises the possibility that checkpoint signaling is still operational at anaphase. It should be noted that there is copious evidence that the checkpoint is inactivated at some point during mitotic exit, either through degradation of checkpoint proteins [14Palframan W.J. Meehl J.B. Jaspersen S.L. Winey M. Murray A.W. Anaphase inactivation of the spindle checkpoint.Science. 2006; 313: 680-684Crossref PubMed Scopus (101) Google Scholar, 24Qi W. Yu H. KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome.J. Biol. Chem. 2007; 282: 3672-3679Crossref PubMed Scopus (60) Google Scholar, 25King E.M. van der Sar S.J. Hardwick K.G. Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint.PLoS ONE. 2007; 2: e342Crossref PubMed Scopus (108) Google Scholar, 26Choi E. Choe H. Min J. Choi J.Y. Kim J. Lee H. BubR1 acetylation at prometaphase is required for modulating APC/C activity and timing of mitosis.EMBO J. 2009; 28: 2077-2089Crossref PubMed Scopus (97) Google Scholar] or through loss of CDK1-dependent phosphorylations [27Chung E. Chen R.H. Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint.Nat. Cell Biol. 2003; 5: 748-753Crossref PubMed Scopus (126) Google Scholar, 28D’Angiolella V. Mari C. Nocera D. Rametti L. Grieco D. The spindle checkpoint requires cyclin-dependent kinase activity.Genes Dev. 2003; 17: 2520-2525Crossref PubMed Scopus (121) Google Scholar, 29Morin V. Prieto S. Melines S. Hem S. Rossignol M. Lorca T. Espeut J. Morin N. Abrieu A. CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint.Curr. Biol. 2012; 22: 289-295Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 30Yamaguchi S. Decottignies A. Nurse P. Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint.EMBO J. 2003; 22: 1075-1087Crossref PubMed Scopus (81) Google Scholar]. However, when with respect to anaphase these mechanisms inactivate the checkpoint is largely unclear. Work from the Petronczki group indicates that recruitment of the checkpoint proteins Mad1 and Mad2 to kinetochores may be impaired by the time of anaphase in human cells [11Vázquez-Novelle M.D. Petronczki M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase.Curr. Biol. 2010; 20: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. Our data suggest that the checkpoint remains operational at anaphase. Yet, in an unperturbed anaphase, rerecruitment of checkpoint proteins to kinetochores is typically not observed (Figures 1A, 1C, and S1E). This indicates that chromosome attachments remain stable, providing no possibility for the checkpoint to become engaged. This also indicates that persistent chromosome attachment is the primary mechanism that ensures unperturbed anaphase progression. Slow checkpoint activation (as we describe here) or an inability to activate the checkpoint in anaphase [11Vázquez-Novelle M.D. Petronczki M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase.Curr. Biol. 2010; 20: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar] will only become functionally important when the primary mechanism fails. This prompted us to ask how likely this is. Stability of chromosome attachment is thought to be regulated by centromere-localized Aurora B [2Carmena M. Wheelock M. Funabiki H. Earnshaw W.C. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis.Nat. Rev. Mol. Cell Biol. 2012; 13: 789-803Crossref PubMed Scopus (563) Google Scholar, 10Mirchenko L. Uhlmann F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Vázquez-Novelle M.D. Petronczki M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase.Curr. Biol. 2010; 20: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. We therefore wanted to know when with respect to anaphase Aurora B translocates from the centromere to the midspindle and how variable this process is. Surprisingly, there was considerable variability and we found Aurora B on centromeres for up to 2 min after anaphase onset (Figures S4A and S4B), consistent with findings in vertebrate cells [31Murata-Hori M. Tatsuka M. Wang Y.L. Probing the dynamics and functions of aurora B kinase in living cells during mitosis and cytokinesis.Mol. Biol. Cell. 2002; 13: 1099-1108Crossref PubMed Scopus (169) Google Scholar]. Hence, attachment remains stable despite the presence of Aurora B on centromeres, strengthening previous hints that an additional mechanism supports chromosome attachment stability in anaphase [11Vázquez-Novelle M.D. Petronczki M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase.Curr. Biol. 2010; 20: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. Like Aurora B translocation [8Pereira G. Schiebel E. Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14.Science. 2003; 302: 2120-2124Crossref PubMed Scopus (220) Google Scholar, 9Hümmer S. Mayer T.U. Cdk1 negatively regulates midzone localization of the mitotic kinesin Mklp2 and the chromosomal passenger complex.Curr. Biol. 2009; 19: 607-612Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar], this other mechanism seems to require declining CDK1/cyclin B activity, because maintaining high cyclin B levels creates unstable chromosome attachments in anaphase (Figures S1B and S1D). Hence, anaphase is at risk as long as cyclin B levels are still (relatively) high. Checkpoint activation on the other hand is slow even when cyclin B levels are high (Figure 3), making it a suitable mechanism to protect anaphase in such a situation. The cells co-overexpressing securin and separase provide a means to test this idea. In these cells, anaphase occurs very early after the APC/C has become active and Aurora B translocates considerably later with respect to anaphase than in wild-type cells (Figure S4C). Hence, there should be a higher tendency for destabilization of chromosome attachments. Consistently, we sometimes see securin stabilization in these cells, indicating that an error was recognized during anaphase (Figure S4D). This needs to be corroborated by visualizing checkpoint proteins, which technical difficulties have so far rendered impossible for us. Most cells proceeded through anaphase unhindered, which suggests that slow checkpoint activation may be efficient in ensuring anaphase progression while cyclin B levels are still high. Since we found slow checkpoint activation kinetics at high cyclin B levels (mimicking the biochemical situation in prometaphase), checkpoint activation may be slow throughout prometaphase. This is surprising because the checkpoint is considered a crucial safety mechanism during that time of mitosis. Yet, this slowness seems evolutionary conserved since the Gerlich group recently reported similarly slow checkpoint activation kinetics during metaphase in human cells [22Dick A.E. Gerlich D.W. Kinetic framework of spindle assembly checkpoint signalling.Nat. Cell Biol. 2013; 15: 1370-1377Crossref PubMed Scopus (122) Google Scholar]. We asked whether such slow checkpoint activation is at all consistent with the timing of mitosis. In an unperturbed S. pombe mitosis, it takes on average 5.8 min from entry into mitosis (when chromosomes are initially unattached) to APC/C activation (Figures S4E and S4F). Apparently, this timing is set by checkpoint-independent mechanisms controlling APC/C activity, because deletions of checkpoint genes do not accelerate mitosis (Figure S4G). For the checkpoint to be able to protect chromosomes from missegregating, attachment errors must block APC/C activity in less than 5.8 min. Our data indicate that it takes on average 4.3 min (in the presence of nondegradable cyclin B; Figure 3F). Hence, there is just enough time for chromosome attachment errors in early mitosis to prevent APC/C activity. In contrast, chromosome attachment errors that occur late in prometaphase may not have enough time to block APC/C activity. This is consistent with observations in human cells [22Dick A.E. Gerlich D.W. Kinetic framework of spindle assembly checkpoint signalling.Nat. Cell Biol. 2013; 15: 1370-1377Crossref PubMed Scopus (122) Google Scholar] and reveals a surprising vulnerability in the checkpoint mechanism. We can envision two possibilities why slow checkpoint activation nevertheless exists and is evolutionary conserved: either there is a biochemical constraint, which makes faster inhibition of the APC/C impossible, or the slowness has been evolutionary conserved because it provides a safety mechanism in anaphase, as our work here suggests. Strains are listed in Table S1. PCR-based gene targeting [32Bähler J. Wu J.Q. Longtine M.S. Shah N.G. McKenzie 3rd, A. Steever A.B. Wach A. Philippsen P. Pringle J.R. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.Yeast. 1998; 14: 943-951Crossref PubMed Scopus (1756) Google Scholar] was used to replace genes by gene fusions at their endogenous loci. For overexpression of securin and separase, we replaced the endogenous promoters by the constitutive Padh1 [33McLeod M. Stein M. Beach D. The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast.EMBO J. 1987; 6: 729-736Crossref PubMed Scopus (187) Google Scholar] and the thiamine-repressible Pnmt1 promoter [34Basi G. Schmid E. Maundrell K. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility.Gene. 1993; 123: 131-136Crossref PubMed Scopus (568) Google Scholar], respectively—except for Figures 2B and 2C, where separase overexpression was achieved by integration of Pnmt1-cut1+-13myc-Tadh1 into the leu1 locus. For inducible expression of ΔN-cdc13, the coding sequence for amino acids 68 to 482 of Cdc13 [16Yamano H. Gannon J. Hunt T. The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.EMBO J. 1996; 15: 5268-5279Crossref PubMed Scopus (136) Google Scholar] was cloned into the pDual vector [35Matsuyama A. Shirai A. Yashiroda Y. Kamata A. Horinouchi S. Yoshida M. pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast.Yeast. 2004; 21: 1289-1305Crossref PubMed Scopus (90) Google Scholar] under control of the Pnmt81 promoter and integrated into the leu1 locus. Unless stated differently, cells were grown at 30°C in Edinburgh minimal medium (EMM) with the necessary supplements. When applicable, the nmt promoter was suppressed by addition of 16 μM thiamine. Protein expression from the nmt promoter and its derivatives at 30°C was induced by culturing of the cells for 14–18 hr in EMM without thiamine. Rich medium (YEA) was used for asynchronously growing cells for protein extraction and immunoblotting. Detailed information can be found in the Supplemental Experimental Procedures. Cells were mounted in lectin-coated (35 μg/ml; Sigma L1395) culture dishes (8-well; Ibidi) and preincubated on the microscope stage for 30 min. Live-cell imaging was carried out at 30°C (if not stated otherwise) on a DeltaVision Core system (Applied Precision/GE Healthcare) equipped with a climate chamber (EMBL) using a 60×/1.4 Apo oil objective (Olympus). Images were deconvolved using SoftWorx software. For representative pictures, maximum-intensity projections were used if z stacks were acquired. All intensity measurements were performed using SoftWorx, and data analysis and kymograph assembly were performed using MatLab. Intensity was measured in units of raw camera counts. A detailed description of the imaging conditions and analysis can be found in the Supplemental Experimental Procedures. J.K. and S.H. conceived the project and wrote the manuscript; J.K. designed, performed, and analyzed experiments. We thank Tomohiro Matsumoto for antibodies, Yoshinori Watanabe for S. pombe strains, Mitsuhiro Yanagida for plasmids, Eva Illgen and Armin Kubis for excellent technical help, members of the Hauf lab for discussions, and Maria Vázquez-Novelle and Mark Petronczki for communicating results prior to publication. This work was supported by the Max Planck Society and the Boehringer Ingelheim Fonds (fellowship to J.K.). Download .pdf (11.15 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Figures S1–S4, and Table S1 Cdk1 Inactivation Terminates Mitotic Checkpoint Surveillance and Stabilizes Kinetochore Attachments in AnaphaseVázquez-Novelle et al.Current BiologyFebruary 27, 2014In BriefVázquez-Novelle et al. propose that Cdk1-cyclin B activity sets the temporal window for microtubule-kinetochore attachment surveillance. Failure to degrade cyclin B at anaphase onset destabilizes attachments and engages the mitotic checkpoint. APC/C-induced proteolysis could couple sister chromatid disjunction to checkpoint dissolution when sister chromatids split. Full-Text PDF Open AccessDependency of the Spindle Assembly Checkpoint on Cdk1 Renders the Anaphase Transition IrreversibleRattani et al.Current BiologyFebruary 27, 2014In BriefRattani et al. show that Cdk1 is required for spindle assembly checkpoint (SAC) activation by unattached/tensionless chromosomes. Cdk1 inactivation through degradation of its cyclin B subunit blocks SAC reactivation during anaphase. The SAC’s dependence on Cdk1 makes separase activation irreversible. Full-Text PDF Open Access" @default.
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• W2043983559 title "Slow Checkpoint Activation Kinetics as a Safety Device in Anaphase" @default.
• W2043983559 cites W1508414663 @default.
• W2043983559 cites W1870919523 @default.
• W2043983559 cites W1963814491 @default.
• W2043983559 cites W1966072213 @default.
• W2043983559 cites W1977345614 @default.
• W2043983559 cites W1990265320 @default.
• W2043983559 cites W1995749082 @default.
• W2043983559 cites W1996481792 @default.
• W2043983559 cites W2018842128 @default.
• W2043983559 cites W2020168080 @default.
• W2043983559 cites W2028511996 @default.
• W2043983559 cites W2032629746 @default.
• W2043983559 cites W2043190827 @default.
• W2043983559 cites W2048938594 @default.
• W2043983559 cites W2050723856 @default.
• W2043983559 cites W2054818623 @default.
• W2043983559 cites W2058582467 @default.
• W2043983559 cites W2059605618 @default.
• W2043983559 cites W2082723977 @default.
• W2043983559 cites W2085170551 @default.
• W2043983559 cites W2091912506 @default.
• W2043983559 cites W2098391208 @default.
• W2043983559 cites W2099558654 @default.
• W2043983559 cites W2102770794 @default.
• W2043983559 cites W2103211352 @default.
• W2043983559 cites W2110718602 @default.
• W2043983559 cites W2113377931 @default.
• W2043983559 cites W2114705132 @default.
• W2043983559 cites W2128608940 @default.
• W2043983559 cites W2138958608 @default.
• W2043983559 cites W2142790777 @default.
• W2043983559 cites W2144863848 @default.
• W2043983559 cites W2159895537 @default.
• W2043983559 cites W2160944075 @default.
• W2043983559 cites W2170145644 @default.
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