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• W2020168080 abstract "The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays anaphase onset until all sister kinetochores have become attached to opposite poles [1Rieder C.L. Schultz A. Cole R. Sluder G. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle.J. Cell Biol. 1994; 127: 1301-1310Crossref PubMed Scopus (429) Google Scholar, 2Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar]. Correct bipolar attachment leads to kinetochore deformation and tension and satisfies the checkpoint [3Li X. Nicklas R.B. Mitotic forces control a cell-cycle checkpoint.Nature. 1995; 373: 630-632Crossref PubMed Scopus (449) Google Scholar, 4Maresca T.J. Salmon E.D. Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity.J. Cell Biol. 2009; 184: 373-381Crossref PubMed Scopus (188) Google Scholar, 5Nezi L. Musacchio A. Sister chromatid tension and the spindle assembly checkpoint.Curr. Opin. Cell Biol. 2009; 21: 785-795Crossref PubMed Scopus (117) Google Scholar, 6Uchida K.S. Takagaki K. Kumada K. Hirayama Y. Noda T. Hirota T. Kinetochore stretching inactivates the spindle assembly checkpoint.J. Cell Biol. 2009; 184: 383-390Crossref PubMed Scopus (177) Google Scholar]. What prevents mitotic checkpoint reactivation when sister centromeres are split and tension is lost at anaphase onset? Aurora B kinase, the catalytic subunit of the chromosomal passenger protein complex (CPC) [7Ruchaud S. Carmena M. Earnshaw W.C. Chromosomal passengers: Conducting cell division.Nat. Rev. Mol. Cell Biol. 2007; 8: 798-812Crossref PubMed Scopus (650) Google Scholar], acts as a sensor at inner centromeres for the status of attachment [5Nezi L. Musacchio A. Sister chromatid tension and the spindle assembly checkpoint.Curr. Opin. Cell Biol. 2009; 21: 785-795Crossref PubMed Scopus (117) Google Scholar, 8Liu D. Vader G. Vromans M.J. Lampson M.A. Lens S.M. Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates.Science. 2009; 323: 1350-1353Crossref PubMed Scopus (386) Google Scholar]. Phosphorylation of Aurora B targets at erroneously attached kinetochores elicits the correction of these attachments and the activation of the mitotic checkpoint. At anaphase, the CPC leaves the centromeres and relocates to the spindle midzone [7Ruchaud S. Carmena M. Earnshaw W.C. Chromosomal passengers: Conducting cell division.Nat. Rev. Mol. Cell Biol. 2007; 8: 798-812Crossref PubMed Scopus (650) Google Scholar]. This iconic translocation might prevent the checkpoint from reengaging after anaphase onset. To test this hypothesis, we experimentally retained Aurora B and the CPC at the centromere throughout anaphase in human cells. Preventing CPC translocation caused the untimely recruitment of mitotic checkpoint proteins to kinetochores at anaphase in an Aurora B kinase activity-dependent manner. Our results suggest that the relocalization of the CPC, an evolutionarily conserved event in eukaryotes, is a key mechanism that incapacitates the mitotic checkpoint at anaphase. The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays anaphase onset until all sister kinetochores have become attached to opposite poles [1Rieder C.L. Schultz A. Cole R. Sluder G. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle.J. Cell Biol. 1994; 127: 1301-1310Crossref PubMed Scopus (429) Google Scholar, 2Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar]. Correct bipolar attachment leads to kinetochore deformation and tension and satisfies the checkpoint [3Li X. Nicklas R.B. Mitotic forces control a cell-cycle checkpoint.Nature. 1995; 373: 630-632Crossref PubMed Scopus (449) Google Scholar, 4Maresca T.J. Salmon E.D. Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity.J. Cell Biol. 2009; 184: 373-381Crossref PubMed Scopus (188) Google Scholar, 5Nezi L. Musacchio A. Sister chromatid tension and the spindle assembly checkpoint.Curr. Opin. Cell Biol. 2009; 21: 785-795Crossref PubMed Scopus (117) Google Scholar, 6Uchida K.S. Takagaki K. Kumada K. Hirayama Y. Noda T. Hirota T. Kinetochore stretching inactivates the spindle assembly checkpoint.J. Cell Biol. 2009; 184: 383-390Crossref PubMed Scopus (177) Google Scholar]. What prevents mitotic checkpoint reactivation when sister centromeres are split and tension is lost at anaphase onset? Aurora B kinase, the catalytic subunit of the chromosomal passenger protein complex (CPC) [7Ruchaud S. Carmena M. Earnshaw W.C. Chromosomal passengers: Conducting cell division.Nat. Rev. Mol. Cell Biol. 2007; 8: 798-812Crossref PubMed Scopus (650) Google Scholar], acts as a sensor at inner centromeres for the status of attachment [5Nezi L. Musacchio A. Sister chromatid tension and the spindle assembly checkpoint.Curr. Opin. Cell Biol. 2009; 21: 785-795Crossref PubMed Scopus (117) Google Scholar, 8Liu D. Vader G. Vromans M.J. Lampson M.A. Lens S.M. Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates.Science. 2009; 323: 1350-1353Crossref PubMed Scopus (386) Google Scholar]. Phosphorylation of Aurora B targets at erroneously attached kinetochores elicits the correction of these attachments and the activation of the mitotic checkpoint. At anaphase, the CPC leaves the centromeres and relocates to the spindle midzone [7Ruchaud S. Carmena M. Earnshaw W.C. Chromosomal passengers: Conducting cell division.Nat. Rev. Mol. Cell Biol. 2007; 8: 798-812Crossref PubMed Scopus (650) Google Scholar]. This iconic translocation might prevent the checkpoint from reengaging after anaphase onset. To test this hypothesis, we experimentally retained Aurora B and the CPC at the centromere throughout anaphase in human cells. Preventing CPC translocation caused the untimely recruitment of mitotic checkpoint proteins to kinetochores at anaphase in an Aurora B kinase activity-dependent manner. Our results suggest that the relocalization of the CPC, an evolutionarily conserved event in eukaryotes, is a key mechanism that incapacitates the mitotic checkpoint at anaphase. CPC relocation prevents mitotic checkpoint engagement at anaphase onset Centromeric CPC retention causes recruitment of Bub1, BubR1, and Mps1 to kinetochores This untimely mitotic checkpoint response at anaphase requires Aurora B activity CPC retention at centromeres does not destabilize K fibers or delay mitotic exit Experiments in Drosophila embryos [9Oliveira 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 (121) Google Scholar] and budding yeast cells (Mirchenko and Uhlmann [10Mirchenko L. Uhlmann F. Sli15INCENP dephosphorylation prevents mitotic checkpoint reactivation due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar], this issue of Current Biology) have revealed that the artificial dissolution of sister chromatid cohesion in metaphase leads to the activation of the mitotic checkpoint in the ensuing pseudo anaphases. In both organisms, this checkpoint response can be suppressed by reversing Cdk1 phosphorylation. In line with previous observations [11Parry 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 (67) Google Scholar], this suggests that in normal anaphase cells, a process controlled by Cdk1 inactivation is responsible for preventing the checkpoint from reengaging during mitotic exit. In yeast [12Pereira G. Schiebel E. Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14.Science. 2003; 302: 2120-2124Crossref PubMed Scopus (214) Google Scholar] and mammalian cells [13Hümmer 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 (112) Google Scholar], the translocation of Aurora B kinase from the inner centromere to the spindle midzone at anaphase is controlled by Cdk1 inactivation and dephosphorylation of the chromosomal passenger protein complex (CPC) subunit INCENP. To test whether the removal of Aurora B from the inner centromere is responsible for preventing the mitotic checkpoint from engaging when sister centromeres are split at anaphase, we first made use of the fact that CPC relocation in mammalian cells requires the kinesin-6 family protein Mklp2 [14Gruneberg U. Neef R. Honda R. Nigg E.A. Barr F.A. Relocation of Aurora B from centromeres to the central spindle at the metaphase to anaphase transition requires MKlp2.J. Cell Biol. 2004; 166: 167-172Crossref PubMed Scopus (217) Google Scholar]. Small interfering RNA (siRNA)-mediated depletion of Mklp2 in HeLa cells (Figure 1B ) did not alter the recruitment of Aurora B to the inner centromere at metaphase but caused the centromeric retention of the protein (Figure 1A) and its CPC partner INCENP in anaphase (Figure 1D). The mitotic checkpoint proteins BubR1 and Bub1 [2Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar] accumulate at kinetochores in prometaphase and largely disappear from chromosomes upon bipolar attachment at metaphase (see Figures S1A and S1B available online). Although both proteins were absent from kinetochores during anaphase in control cells, BubR1 and Bub1 were recruited to anaphase kinetochores in Mklp2-depleted cells (Figures 1C and 1D). Depletion of the second human kinesin-6 family protein, Mklp1, which, like Mklp2, localizes to the spindle midzone and is required for cytokinesis [15Glotzer M. The 3Ms of central spindle assembly: Microtubules, motors and MAPs.Nat. Rev. Mol. Cell Biol. 2009; 10: 9-20Crossref PubMed Scopus (256) Google Scholar], neither prevented CPC relocation nor caused untimely localization of mitotic checkpoint proteins to kinetochores during anaphase (Figures 1C and 1D). Crucially, acute inhibition of Aurora B at anaphase by treatment with ZM447439 [16Ditchfield C. Johnson V.L. Tighe A. Ellston R. Haworth C. Johnson T. Mortlock A. Keen N. Taylor S.S. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.J. Cell Biol. 2003; 161: 267-280Crossref PubMed Scopus (1007) Google Scholar] abolished the kinetochore localization of both BubR1 and Bub1 in Mklp2-depleted cells (Figures 2A and 2B ), suggesting that this effect is dependent on the kinase activity of Aurora B.Figure 2Preventing Chromosomal Passenger Protein Complex Relocation Triggers Aurora B-Dependent Recruitment of the Mitotic Checkpoint Proteins BubR1 and Bub1 to Anaphase KinetochoresShow full caption(A) Aurora B and BubR1 localization was analyzed by IF in control and Mklp2-depleted cells synchronously released into anaphase and treated with solvent control dimethyl sulfoxide (DMSO) or the Aurora B inhibitor ZM447439 (n > 41). Please note that inhibition of Aurora B alters the subcellular localization of chromosomal passenger protein complex (CPC) proteins.(B) INCENP and Bub1 localization was analyzed by IF in cells treated as in (A) (n > 94).(C) Immunoblot analysis of extracts prepared from cells 39 hr after transfection with no DNA (control), a plasmid encoding AcGFP-INCENPWT, and a plasmid encoding AcGFP-INCENPT59E. The blots were probed with antibodies directed against INCENP (top), AcGFP (middle), and α-tubulin (bottom).(D) AcGFP-INCENP and BubR1 localization was analyzed by IF in cells transiently transfected with plasmids encoding AcGFP-INCENPWT and AcGFP-INCENPT59E (n > 26).(E) AcGFP-INCENP and Bub1 localization was analyzed by IF cells transiently transfected with a plasmid encoding AcGFP-INCENPT59E. Cells were synchronously released into anaphase and treated with solvent control DMSO or the Aurora B inhibitor ZM447439 (n = 40). Scale bars represent 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Aurora B and BubR1 localization was analyzed by IF in control and Mklp2-depleted cells synchronously released into anaphase and treated with solvent control dimethyl sulfoxide (DMSO) or the Aurora B inhibitor ZM447439 (n > 41). Please note that inhibition of Aurora B alters the subcellular localization of chromosomal passenger protein complex (CPC) proteins. (B) INCENP and Bub1 localization was analyzed by IF in cells treated as in (A) (n > 94). (C) Immunoblot analysis of extracts prepared from cells 39 hr after transfection with no DNA (control), a plasmid encoding AcGFP-INCENPWT, and a plasmid encoding AcGFP-INCENPT59E. The blots were probed with antibodies directed against INCENP (top), AcGFP (middle), and α-tubulin (bottom). (D) AcGFP-INCENP and BubR1 localization was analyzed by IF in cells transiently transfected with plasmids encoding AcGFP-INCENPWT and AcGFP-INCENPT59E (n > 26). (E) AcGFP-INCENP and Bub1 localization was analyzed by IF cells transiently transfected with a plasmid encoding AcGFP-INCENPT59E. Cells were synchronously released into anaphase and treated with solvent control DMSO or the Aurora B inhibitor ZM447439 (n = 40). Scale bars represent 10 μm. To confirm that the centromeric retention of Aurora B and the CPC is responsible for mitotic checkpoint protein accumulation at anaphase kinetochores, we transiently expressed INCENP carrying a T59E substitution that mimics constitutive phosphorylation by Cdk1 [13Hümmer 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 (112) Google Scholar] (Figure 2C). Whereas ectopically expressed INCENPWT translocated to the spindle midzone at anaphase onset, INCENPT59E persisted at centromeres (Figure 2D). This retention of INCENPT59E at centromeres was accompanied by the recruitment of BubR1 and Bub1 to anaphase kinetochores in an Aurora B activity-dependent manner (Figures 2D and 2E; data not shown). Thus, dephosphorylation of INCENP at anaphase and the concomitant relocation of the CPC prevents kinetochore recruitment of mitotic checkpoint proteins. The BubR1 and Bub1 signals in Mklp2-depleted and INCENPT59E-expressing anaphase cells could reflect Aurora B-dependent persistence of the low levels of the checkpoint proteins at kinetochores that are observed at metaphase (Figures S1A and S1B). Alternatively, the loss of sister chromatid cohesion and tension at kinetochores could lead to changes in kinetochore geometry at anaphase onset that are detected by Aurora B. To distinguish between these two possibilities, we quantified the intensity of BubR1 and Bub1 kinetochore staining at different mitotic stages (Figure S2). In both control and Mklp2-depleted cells, the kinetochore intensity of the two checkpoint proteins peaked at prometaphase and strongly declined at metaphase (Figure 3A). Whereas the kinetochore intensity of BubR1 and Bub1 declined further to almost undetectable levels at anaphase in control cells, it increased approximately 4-fold from metaphase to anaphase in Mklp2-depleted cells (Figure 3A). This anaphase-specific surge in BubR1 and Bub1 recruitment upon centromeric retention of the CPC suggests that a change in kinetochore geometry or loss of tension is detected by Aurora B. This conclusion is further supported by the analysis of a third mitotic checkpoint protein, the kinase Mps1 [2Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar]. Mps1 localizes to kinetochores prior to biorientation but is largely lost from kinetochores at metaphase (Figure S1E). Although Mps1 remained absent from kinetochores during anaphase in control cells, the protein was recruited to anaphase kinetochores in Mklp2-depleted cells (Figure 3B). As in the case of BubR1 and Bub1 (Figure 2), this effect was abolished by acute treatment with the Aurora B inhibitor ZM447439 (data not shown). Thus, centromeric retention of the CPC is sufficient to promote the recruitment of three mitotic checkpoint proteins to kinetochores at anaphase. The mitotic checkpoint controls anaphase onset by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that induces the degradation of the anaphase inhibitor securin and cyclin B to allow mitotic exit [2Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar]. Untimely mitotic checkpoint reactivation and subsequent stabilization of cyclin B levels at anaphase could delay or block mitotic exit. To address whether preventing CPC relocation from centromeres to the spindle midzone inhibits the activity of APC, we measured the degradation kinetics of cyclin B in live cells. Neither the rate nor efficiency of cyclin B degradation was significantly impaired in Mklp2-depleted cells when compared to control cells (Figure 4A ; Movie S1). Consistent with this observation, reformation and closure of the nuclear envelope, two events that mark mitotic exit, were not delayed in Mklp2-depleted cells (Figure S3; Movie S2). Thus, the retention of Aurora B at centromeres and recruitment of BubR1, Bub1, and Mps1 to anaphase kinetochores does not suffice to inhibit the APC and block mitotic exit. The corollary of this is that events in addition to the relocation of the CPC suppress the mitotic checkpoint response when sister centromeres are split at anaphase. To address this, we focused on Mad2 and Mad1 [2Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar], two mitotic checkpoint proteins that are recruited to prometaphase kinetochores but dissociate upon bipolar attachment at metaphase (Figures S1C and S1D). In contrast to the aforementioned checkpoint proteins, Mad2 and Mad1 did not accumulate at kinetochores in Mklp2-depleted anaphase in cells (Figures 4B and 4C). This discrepancy suggests that retaining the CPC at centromeres during anaphase elicits a partial checkpoint response. Furthermore, the lack of Mad2 and Mad1 recruitment could explain why APC activity and mitotic exit are not inhibited in anaphase cells that retain Aurora B on centromeres. Aurora B corrects erroneously attached kinetochores by destabilizing microtubule-kinetochore attachments [17Hauf S. Cole R.W. LaTerra S. Zimmer C. Schnapp G. Walter R. Heckel A. van Meel J. Rieder C.L. Peters J.M. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.J. Cell Biol. 2003; 161: 281-294Crossref PubMed Scopus (943) Google Scholar, 18Tanaka T.U. Rachidi N. Janke C. Pereira G. Galova M. Schiebel E. Stark M.J. Nasmyth K. Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections.Cell. 2002; 108: 317-329Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar]. To test whether centromeric retention of the CPC destabilizes attachments when sister chromatids part at anaphase, we analyzed kinetochore fiber (K fiber) stability by cold treatment. This treatment was able to differentiate between cold-resistant attachments in MG132-arrested metaphase cells and unstable microtubules in cells that were treated with the Plk1 inhibitor BI 2536 [19Lénárt P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar] (Figure 4D). Importantly, bundles of cold-resistant K fibers were detected in both control and Mklp2-depleted anaphase cells (Figure 4D). In addition, the absence of Mklp2 did not increase the incidence of lagging chromosomes at anaphase (Figure 4B). These experiments suggest that kinetochore attachments in anaphase cells are stable despite the presence of centromeric Aurora B. Because Mad2 is known to localize to unattached kinetochores [2Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar], the results of the K fiber analysis raised the possibility that the protein is unable to localize to these structures in Mklp2-depleted anaphase cells, because no unattached kinetochores are generated. To test this hypothesis, we acutely depolymerized microtubules in control and Mklp2-depleted cells at different mitotic stages using nocodazole (Figure S4). Mad2 was efficiently recruited to kinetochores upon microtubule depolymerization in MG132-arrested metaphase cells (Figure S4B). In contrast, Mad2 failed to accumulate at kinetochores in control and Mklp2-depleted cells at anaphase following acute nocodazole treatment (Figure S4C). Bub1 recruitment to kinetochores in Mklp2-depleted cells was not affected by microtubule depolymerization (Figure S4C). Thus, Mad2 is unable to localize to entirely unattached kinetochores at anaphase. Collectively, our K fiber stability and microtubule depolymerization analyses suggest that the destabilization of tensionless attachments and the ability of Mad2 to localize to kinetochores are independently suppressed after anaphase onset. Both processes could be differentially controlled before and after the metaphase-to-anaphase transition by the activation state of Cdk1. Recent studies have suggested that kinetochore stretching and tension remove Aurora B substrates from the enzyme's reach when bipolar attachment is achieved [8Liu D. Vader G. Vromans M.J. Lampson M.A. Lens S.M. Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates.Science. 2009; 323: 1350-1353Crossref PubMed Scopus (386) Google Scholar]. This change in kinetochore geometry could allow Aurora B to differentiate between erroneous and bipolar attachments. At anaphase, splitting of sister centromeres and consecutive loss of tension could lead to the relaxation of kinetochore stretching [20Joglekar A.P. Bloom K. Salmon E.D. In vivo protein architecture of the eukaryotic kinetochore with nanometer scale accuracy.Curr. Biol. 2009; 19: 694-699Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar]. This could trigger the mitotic checkpoint and error correction pathway. Consistent with this notion, artificial dissolution of sister chromatid cohesion at metaphase leads to mitotic checkpoint activation and destabilization of attachments in Drosophila embryos [9Oliveira 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 (121) Google Scholar] and to checkpoint activation in budding yeast [10Mirchenko L. Uhlmann F. Sli15INCENP dephosphorylation prevents mitotic checkpoint reactivation due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. Cdk1 inactivation in flies and a mitotic exit pathway in yeast involving separase and Cdc14, a phosphatase that antagonizes Cdk1, suppress this checkpoint response. This suggests that rendering anaphase cells refractory to loss of tension at anaphase onset is a conserved requirement in eukaryotes that is controlled by Cdk1 inactivation. We show here that Cdk1-controlled relocation of the CPC is a key mechanism that prevents mitotic checkpoint engagement at anaphase in human cells. Centromeric retention of the CPC leads to Aurora B activity-dependent and anaphase-specific recruitment of the mitotic checkpoint proteins BubR1, Bub1, and Mps1 to kinetochores. Consistent with our data in human cells, activation of the mitotic checkpoint in artificially induced anaphases in yeast requires the chromosomal association of the Aurora kinase ortholog Ipl1 [10Mirchenko L. Uhlmann F. Sli15INCENP dephosphorylation prevents mitotic checkpoint reactivation due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. Our results also have implications for how the mitotic checkpoint operates in early mitosis. Previous studies have revealed that, prior to anaphase onset, Aurora B is required for BubR1 localization to kinetochores [16Ditchfield C. Johnson V.L. Tighe A. Ellston R. Haworth C. Johnson T. Mortlock A. Keen N. Taylor S.S. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.J. Cell Biol. 2003; 161: 267-280Crossref PubMed Scopus (1007) Google Scholar] and for kinetochore recruitment of Bub1 in response to a decline in tension [21Famulski J.K. Chan G.K. Aurora B kinase-dependent recruitment of hZW10 and hROD to tensionless kinetochores.Curr. Biol. 2007; 17: 2143-2149Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. We show that retaining Aurora B at centromeres is sufficient for the recruitment of BubR1, Bub1, and Mps1, suggesting that the kinase directly controls the association of these checkpoint components with kinetochores independently of Cdk1 activity. Our data are consistent with the role of Aurora B as a sensor that responds to loss of tension by recruiting these three checkpoint proteins. Cells retaining the CPC at anaphase centromeres contain stable K fibers, but they accumulate mitotic checkpoint proteins at anaphase onset. This addresses an important question in the field and suggests that the mitotic checkpoint machinery is capable of responding to a loss of tension even in the presence of stable microtubule-kinetochore attachments. Although centromeric retention of the CPC at anaphase causes hallmarks of mitotic checkpoint activation, it does not suffice to mount a full response. Studies in Xenopus extracts revealed that Cdk1 activity is required for the inhibition of the APC by the mitotic checkpoint [22D'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 (118) Google Scholar]. Our results show that even in cells retaining the CPC at centromeres, the localization of Mad2 to kinetochores and the destabilization of attachments are suppressed after anaphase onset. Supported by evidence in Xenopus [23Wong O.K. Fang G. Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores.J. Cell Biol. 2005; 170: 709-719Crossref PubMed Scopus (55) Google Scholar], Drosophila [9Oliveira 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 (121) Google Scholar], and yeast [10Mirchenko L. Uhlmann F. Sli15INCENP dephosphorylation prevents mitotic checkpoint reactivation due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar], it is tempting to speculate that both processes are activated by Cdk1 in early mitosis. The mechanisms that underlie this temporal regulation and that act in parallel to the Cdk1-controlled relocation of the CPC to suppress the mitotic checkpoint at anaphase have to be the focus of future investigation. In animal cells, the translocation of the CPC to the spindle midzone is required for cytokinesis [13Hümmer 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 (112) Google Scholar]. Based on the results obtained in budding yeast [10Mirchenko L. Uhlmann F. Sli15INCENP dephosphorylation prevents mitotic checkpoint reactivation due to loss of tension at anaphase onset.Curr. Biol. 2010; 20: 1396-1401Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar] and our data in human cells, we propose that Aurora B's departure from chromosomes, which is controlled by INCENP dephosphorylation, is a conserved mechanism that incapacitates the mitotic checkpoint when sister centromeres are split at anaphase onset. HeLa “Kyoto” cells were grown as described in [24Petronczki M. Glotzer M. Kraut N. Peters J.M. Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle.Dev. Cell. 2007; 12: 713-725Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar]. The medium was supplemented with 500 μg/ml G418 for HeLa Mad2-EGFP [25Hutchins J.R. Toyoda Y. Hegemann B. Poser I. Hériché J.K. Sykora M.M. Augsburg M. Hudecz O. Buschhorn B.A. Bulkescher J. et al.Systematic analysis of human protein complexes identifies chromosome segregation proteins.Science. 2010; 328: 593-599Crossref PubMed Scopus (380) Google Scholar] and 200 μg/ml hygromycin B for mCherry-Cyclin B1 [6Uchida K.S. Takagaki K. Kumada K. Hirayama Y. Noda T. Hirota T. Kinetochore stretching inactivates the spindle assembly checkpoint.J. Cell Biol. 2009; 184: 383-390Crossref PubMed Scopus (177) Google Scholar] HeLa “Kyoto” cells. Cells were transfected using Lipofectamine RNAiMax (Invitrogen) with stealth siRNA duplexes (Invitrogen) targeting Mklp1 (KIF23HSS114138), Mklp2 (KIF20AHSS115374), or negative universal control siRNA medium GC at a final concentration of 20 nM. siRNA duplexes were transfected into cycling cells 24 hr (Figure 1A) and 48 hr (Figure 1B) before analysis, respectively. Alternatively, cells were transfected immediately prior to synchronization with 2 mM thymidine (Sigma) for 22 hr. Thymidine-arrested cells were released for 9 hr before fixation (Figures 1C and 1D; Figure 2D; Figures 3A and 3B; Figures 4B–4D; Figure S2; Figure S4) or for 8 hr before recording (Figure 4A; Figure S3), or they were released and arrested in mitosis using MG132 as described [24Petronczki M. Glotzer M. Kraut N. Peters J.M. Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle.Dev. Cell. 2007; 12: 713-725Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar]. For experiments described in Figures 2A, 2B, and 2E, cells were released from MG132 (Sigma), treated with dimethyl sulfoxide or 4 μM Aurora B kinase inhibitor ZM447439 (Tocris) [16Ditchfield C. Johnson V.L. Tighe A. Ellston R. Haworth C. Johnson T. Mortlock A. Keen N. Taylor S.S. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.J. Cell Biol. 2003; 161: 267-280Crossref PubMed Scopus (1007) Google Scholar] 40 min after release, and fixed 70 or 90 min after release. Cycling cells were transfected using FuGene 6 (Roche) with plasmids pIRESpuro3-AcGFP-INCENPWT or pIRESpuro3-AcGFP-INCENPT59E 24 hr before treatment with 50 ng/ml nocodazole (Sigma) for 15 hr (Figure 2C) or immediately before cells were arrested using thymidine (Figures 2D and 2E). Cells were transfected with pIRES-puro3-IBB-DiHcRed1 (based on [26Dultz E. Zanin E. Wurzenberger C. Braun M. Rabut G. Sironi L. Ellenberg J. Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells.J. Cell Biol. 2008; 180: 857-865Crossref PubMed Scopus (176) Google Scholar]) 12 hr before treatment with thymidine and siRNA transfection (Figure S3). Cells were treated with 10 μM MG132 or 100 nM BI 2536 [19Lénárt P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar] for 2 hr (Figure 4D; Figure S4B) and with 1 μg/ml nocodazole (Sigma) for 10 min (Figure S4) prior to fixation. Primary antibodies used for immunofluorescence were mouse monoclonal anti-α-tubulin (B512, Sigma, 1:500 for Figure 4D and 1:40,000 for Figure S4A), combined rat monoclonal anti-α-tubulin (clones YLI/2 and YOL1/34, Serotec, 1:2000 each), mouse monoclonal anti-aurora B (AIM-1, BD Transduction Laboratories, 1:500), mouse monoclonal anti-Bub1 (clone14H5, MBL International, 1:200), sheep anti-BubR1 ([27Taylor S.S. Hussein D. Wang Y. Elderkin S. Morrow C.J. Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells.J. Cell Sci. 2001; 114: 4385-4395Crossref PubMed Google Scholar], 1:1000), human CREST anti-centromere (Inmunovision, 1:4000), mouse monoclonal anti-GFP (clones 7.1 and 13.1, Roche, 1:1000), rabbit polyclonal anti-INCENP ([17Hauf S. Cole R.W. LaTerra S. Zimmer C. Schnapp G. Walter R. Heckel A. van Meel J. Rieder C.L. Peters J.M. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.J. Cell Biol. 2003; 161: 281-294Crossref PubMed Scopus (943) Google Scholar], 1:1500), Alexa 488-conjugated mouse monoclonal anti-Mad1 (clone BB3-8 [28Vink M. Simonetta M. Transidico P. Ferrari K. Mapelli M. De Antoni A. Massimiliano L. Ciliberto A. Faretta M. Salmon E.D. Musacchio A. In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.Curr. Biol. 2006; 16: 755-766Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar], 1:600), and mouse monoclonal anti-Mps1 (clone N1, Invitrogen, 1:500). Cross-adsorbed secondary antibodies conjugated to Alexa 488 or Alexa 594 (Molecular Probes, Invitrogen) were used for detection. Primary antibodies used for immunoblotting were mouse monoclonal anti-α-tubulin (B512, Sigma, 1:75,000), mouse monoclonal anti-AcGFP (JL-8, Living Colors, 1:5000), mouse monoclonal anti-GAPDH (clone 6C5, Abcam, 1:30,000), rabbit polyclonal anti-INCENP (ab2183, AbCam, 1:2000), mouse monoclonal anti-Mklp1 (clone 24, BD Transduction Laboratories, 1:1000), and sheep anti-Mklp2 (FBA38 [29Neef R. Preisinger C. Sutcliffe J. Kopajtich R. Nigg E.A. Mayer T.U. Barr F.A. Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis.J. Cell Biol. 2003; 162: 863-875Crossref PubMed Scopus (249) Google Scholar], 1:4000). HeLa “Kyoto” and HeLa Mad2-EGFP were fixed with 4% formaldehyde and processed for immunofluorescence microscopy (IF) as described [19Lénárt P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar]. Alternatively, cells were fixed with −20°C methanol (Figure 1A; Figure S4A) or with 4% formaldehyde in PHEM buffer containing 0.2% Triton X-100 (Figure 3B). Cold treatment and K fiber analysis were performed according to [19Lénárt P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar]. Images were acquired on a Zeiss Axio Imager M1 microscope using a Plan Apochromat 63×/1.4 oil objective lens (Zeiss) equipped with an ORCA-ER camera (Hamamatsu) and controlled by Volocity 4.3.2 software (Improvision). Images are displayed as maximum-intensity projections of deconvolved Z planes (generated by Volocity's iterative restoration function) that were acquired in 0.1 μm sections. IF quantification images were acquired as 12-bit images at identical exposure and processed using ImageJ 1.42q. BubR1 and Bub1 kinetochore mean intensities were measured in nondeconvolved single Z plane images in a circular region with a fixed 9 pixel diameter surrounding the CREST signal. Frames were acquired using a 40× EC Plan-Neofluor 40×/1.30 oil DICII objective lens (Figure 4A; Movie S1) or Plan-Apochromat 10×/0.45 objective (Figure S3; Movie S2) on an Axio Observer Z1 microscope (Zeiss) controlled by SimplePCI software (Hamamatsu), equipped with a full-enclosure environmental chamber heated to 37°C (Digital Pixel Imaging) and an Orca 03GO1 camera (Hamamatsu). Frames were recorded as three Z planes (5 μm apart) every 2 min for the duration of 12 hr (Figure 4A; Movie S1) or as a single Z plane every 3 min for the duration of 10 hr (Figure S3; Movie S2). For quantification, images were acquired as raw 12-bit images and were processed using ImageJ 1.42q. The integrated cellular intensity of mCherry-Cyclin B1 was determined using the in-focus Z plane image at each time point, followed by subtraction of the background signal outside of the cell. We would like to thank Francis Barr, Bill Earnshaw, Toru Hirota, Tony Hyman, Peter Lénart, Prasad Jallepalli, Andrea Musacchio, Jan-Michael Peters, Ted Salmon, and Stephen Taylor for reagents and advice. We would like to acknowledge Lesia Mirchenko and Frank Uhlmann for helpful discussions and for communicating results prior to publication. This work is supported by Cancer Research UK and a Ramón Areces Foundation fellowship to M.D.V.-N. Download .pdf (.69 MB) Help with pdf files Document S1. Four Figures Download .mov (6.76 MB) Help with mov files Movie S1. Time-Lapse Analysis of mCherry-Cyclin B1 Degradation in Control and Mklp2-Depleted Cells (Related to Figure 4)Time point t = 0 is set to the beginning of continuous Cyclin B1 degradation. Download .mov (3.17 MB) Help with mov files Movie S2. Time-Lapse Analysis of IBB-DiHcRed1 Nuclear Import in Control and Mklp2-Depleted Cells (Related to Figure 4 and Figure S3)Time point t = 0 is set to anaphase onset. Sli15INCENP Dephosphorylation Prevents Mitotic Checkpoint Reengagement Due to Loss of Tension at Anaphase OnsetMirchenko et al.Current BiologyJuly 8, 2010In BriefThe mitotic checkpoint, also known as the spindle assembly checkpoint, delays anaphase onset until all chromosomes have reached bipolar tension on the mitotic spindle [1–3]. Once this is achieved, the protease separase is activated to cleave the chromosomal cohesin complex, thereby triggering anaphase. Cohesin cleavage releases tension between sister chromatids, but why the mitotic checkpoint now remains silent is poorly understood. Here, using budding yeast as a model, we show that loss of sister chromatid cohesion at anaphase onset would engage the mitotic checkpoint if this was not prevented by concomitant separase-dependent activation of the Cdc14 phosphatase. Full-Text PDF Open Access" @default.
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• W2020168080 title "Relocation of the Chromosomal Passenger Complex Prevents Mitotic Checkpoint Engagement at Anaphase" @default.
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