FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2787103295> ?p ?o ?g. }

• W2787103295 endingPage "1438" @default.
• W2787103295 startingPage "1424" @default.
• W2787103295 abstract "•SAC-deficient HAP1 cells are viable•15 genes, including BUB1, are synthetic lethal with loss of MAD1 and MAD2•BUB1 is essential for chromosome congression but not for the SAC•The C-terminal tail of BUB1 regulates chromosome congression The spindle assembly checkpoint (SAC) ensures faithful segregation of chromosomes. Although most mammalian cell types depend on the SAC for viability, we found that human HAP1 cells can grow SAC independently. We generated MAD1- and MAD2-deficient cells and mutagenized them to identify synthetic lethal interactions, revealing that chromosome congression factors become essential upon SAC deficiency. Besides expected hits, we also found that BUB1 becomes essential in SAC-deficient cells. We found that the BUB1 C terminus regulates alignment as well as recruitment of CENPF. Second, we found that BUBR1 was not essential in SAC-deficient HAP1 cells. We confirmed that BUBR1 does not regulate chromosome alignment in HAP1 cells and that BUB1 does not regulate chromosome alignment through BUBR1. Taken together, our data resolve some long-standing questions about the interplay between BUB1 and BUBR1 and their respective roles in the SAC and chromosome alignment. The spindle assembly checkpoint (SAC) ensures faithful segregation of chromosomes. Although most mammalian cell types depend on the SAC for viability, we found that human HAP1 cells can grow SAC independently. We generated MAD1- and MAD2-deficient cells and mutagenized them to identify synthetic lethal interactions, revealing that chromosome congression factors become essential upon SAC deficiency. Besides expected hits, we also found that BUB1 becomes essential in SAC-deficient cells. We found that the BUB1 C terminus regulates alignment as well as recruitment of CENPF. Second, we found that BUBR1 was not essential in SAC-deficient HAP1 cells. We confirmed that BUBR1 does not regulate chromosome alignment in HAP1 cells and that BUB1 does not regulate chromosome alignment through BUBR1. Taken together, our data resolve some long-standing questions about the interplay between BUB1 and BUBR1 and their respective roles in the SAC and chromosome alignment. The spindle assembly checkpoint (SAC; also known as the mitotic checkpoint) prevents errors in chromosome segregation. Components of the SAC localize to unattached kinetochores (KTs), where they generate a diffusible signal, known as the mitotic checkpoint complex (MCC). The MCC is composed of MAD2, BUBR1, and BUB3, which together prevent anaphase onset by binding to CDC20 and thereby preventing activation of the anaphase-promoting complex/cyclosome (APC/C) (Foley and Kapoor, 2013Foley E.A. Kapoor T.M. Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore.Nat. Rev. Mol. Cell Biol. 2013; 14: 25-37Crossref PubMed Scopus (454) Google Scholar). In addition, MAD1, which requires BUB1, NDC80, and the RZZ complex to localize to unattached kinetochores, acts as a “hub” at unattached kinetochores to catalyze the formation of the MCC (Stukenberg and Burke, 2015Stukenberg P.T. Burke D.J. Connecting the microtubule attachment status of each kinetochore to cell cycle arrest through the spindle assembly checkpoint.Chromosoma. 2015; 124: 463-480Crossref PubMed Scopus (30) Google Scholar). Upon full chromosome alignment, the SAC is silenced and cells progress into anaphase. Some less divergent eukaryotes, such as yeast and flies, can survive without a functional SAC, as they accomplish chromosome alignment very efficiently. In contrast, the SAC appears to be essential for viability in higher organisms (Dobles et al., 2000Dobles M. Liberal V. Scott M.L. Benezra R. Sorger P.K. Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2.Cell. 2000; 101: 635-645Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, Kalitsis et al., 2000Kalitsis P. Earle E. Fowler K.J. Choo K.H. Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis.Genes Dev. 2000; 14: 2277-2282Crossref PubMed Scopus (216) Google Scholar, Michel et al., 2001Michel L.S. Liberal V. Chatterjee A. Kirchwegger R. Pasche B. Gerald W. Dobles M. Sorger P.K. Murty V.V. Benezra R. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells.Nature. 2001; 409: 355-359Crossref PubMed Scopus (645) Google Scholar, Perera et al., 2007Perera D. Tilston V. Hopwood J.A. Barchi M. Boot-Handford R.P. Taylor S.S. Bub1 maintains centromeric cohesion by activation of the spindle checkpoint.Dev. Cell. 2007; 13: 566-579Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In cell culture, the SAC was found to be essential for a plethora of different transformed cell lines (Kops et al., 2004Kops G.J. Foltz D.R. Cleveland D.W. Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint.Proc. Natl. Acad. Sci. USA. 2004; 101: 8699-8704Crossref PubMed Scopus (346) Google Scholar, Michel et al., 2004Michel L. Diaz-Rodriguez E. Narayan G. Hernando E. Murty V.V.V.S. Benezra R. Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells.Proc. Natl. Acad. Sci. USA. 2004; 101: 4459-4464Crossref PubMed Scopus (168) Google Scholar). In contrast, MAD2 null mouse embryonic fibroblasts can be established in a p53-deficient background (Burds et al., 2005Burds A.A. Lutum A.S. Sorger P.K. Generating chromosome instability through the simultaneous deletion of Mad2 and p53.Proc. Natl. Acad. Sci. USA. 2005; 102: 11296-11301Crossref PubMed Scopus (93) Google Scholar), and studies in mice showed that loss of MAD2 is tolerated in skin epidermal cells, hepatocytes, and adult T cells (Foijer et al., 2013Foijer F. DiTommaso T. Donati G. Hautaviita K. Xie S.Z. Heath E. Smyth I. Watt F.M. Sorger P.K. Bradley A. Spindle checkpoint deficiency is tolerated by murine epidermal cells but not hair follicle stem cells.Proc. Natl. Acad. Sci. USA. 2013; 110: 2928-2933Crossref PubMed Scopus (40) Google Scholar, Foijer et al., 2017Foijer F. Albacker L.A. Bakker B. Spierings D.C. Yue Y. Xie S.Z. Davis S. Lutum-Jehle A. Takemoto D. Hare B. et al.Deletion of the MAD2L1 spindle assembly checkpoint gene is tolerated in mouse models of acute T-cell lymphoma and hepatocellular carcinoma.eLife. 2017; 6: 341Crossref Scopus (41) Google Scholar). Based on a genome-wide screen for gene essentiality in human haploid HAP1 cells (Blomen et al., 2015Blomen V.A. Májek P. Jae L.T. Bigenzahn J.W. Nieuwenhuis J. Staring J. Sacco R. van Diemen F.R. Olk N. Stukalov A. et al.Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350: 1092-1096Crossref PubMed Scopus (505) Google Scholar), we predicted that loss of the SAC can be tolerated in cultured HAP1 cells. Indeed, we were able to generate MAD1 and MAD2 knockout cell lines in the HAP1 background. As the SAC becomes critically important in a context where chromosome alignment is perturbed or delayed, we used these cells to find specific regulators of this process that are synthetic lethal with SAC deficiency. Although MAD1 and MAD2 are required for the proper functioning of the SAC, both were classified as nonessential in HAP1 cells (Blomen et al., 2015Blomen V.A. Májek P. Jae L.T. Bigenzahn J.W. Nieuwenhuis J. Staring J. Sacco R. van Diemen F.R. Olk N. Stukalov A. et al.Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350: 1092-1096Crossref PubMed Scopus (505) Google Scholar). Indeed, we could easily obtain MAD1 and MAD2 knockout cell lines using a RNA-guided Cas9-nuclease-based genome editing strategy (Figures 1A, 1B, and S1A–S1C). As expected, both the ΔMAD1 and ΔMAD2 cell lines failed to delay mitosis in response to the spindle poison nocodazole, and the duration of mitosis was decreased (Figures 1C and 1D). Also, cyclin B1 degradation was triggered immediately after nuclear envelope breakdown (NEB) in ΔMAD2 cells, irrespective of the status of chromosome alignment (Figures S2A and S2B). Despite the reduced time spent in mitosis, ΔMAD1 and ΔMAD2 cells displayed only a slight increase in chromosome segregation errors (Figure 1E), indicating that HAP1 cells complete chromosome alignment relatively efficiently, allowing growth in a SAC-independent manner. This is not due to the fact that HAP1 cells have half the number of chromosomes, since diploid HAP1-derived lines of the ΔMAD2 genotype were also viable and the rates of spontaneous chromosome segregation errors were not increased as compared to their haploid counterpart (Figures S2C–S2E). However, the ΔMAD1 and ΔMAD2 cell lines were more sensitive to low doses of spindle poisons, such as Taxol or nocodazole (Figure 1F), indicating that these cells are more sensitive to perturbations that delay the process of chromosome alignment. The increased sensitivity of SAC-deficient cells to spindle perturbations encouraged us to screen for genes that are specifically essential for viability of SAC-deficient cell lines. Such screens are expected to identify genes that promote chromosome congression, but not those that are absolutely required for kinetochore-microtubule attachments, as these are also expected to be essential in the WT HAP1 cells. We performed insertional mutagenesis screens using a gene trap virus in two independent ΔMAD1 clones and two independent ΔMAD2 clones. Considering the gene trap is unidirectional by design, the proportion of integrations recovered in the transcriptional orientation of a gene can function as a measure of mutant fitness (Figure 2A; Blomen et al., 2015Blomen V.A. Májek P. Jae L.T. Bigenzahn J.W. Nieuwenhuis J. Staring J. Sacco R. van Diemen F.R. Olk N. Stukalov A. et al.Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350: 1092-1096Crossref PubMed Scopus (505) Google Scholar). We found 15 genes to be specifically required for fitness in ΔMAD1 and ΔMAD2 cells (Figures 2B–2E). Importantly, there was an enrichment for kinetochore-associated genes, among which several known regulators of chromosome alignment; CENPE, ROD (an RZZ-complex component), ZWINT, EB1, CCNB1, PPP6R3, and CENPP and CENPO (two members of a five subunit subcomplex of the CCAN network) (see Supplemental References for prior work on these proteins). We also identified all subunits of the condensin II complex. Although condensin II has been implicated in chromosome segregation (Hirano, 2016Hirano T. Condensin-based chromosome organization from bacteria to vertebrates.Cell. 2016; 164: 847-857Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), its exact role in chromosome alignment is not clear. To validate our hits, we first generated three independent CENPE knockout HAP1 clones (Figures S1D, S3B, and S3C), since CENPE is a known chromosome congression factor and a prominent hit in our screen (Figures 2B–2E and S3A). ΔCENPE cells were completely resistant to GSK-923295, an allosteric inhibitor of CENPE (Qian et al., 2010Qian X. McDonald A. Zhou H.-J. Adams N.D. Parrish C.A. Duffy K.J. Fitch D.M. Tedesco R. Ashcraft L.W. Yao B. et al.Discovery of the First Potent and Selective Inhibitor of Centromere-Associated Protein E: GSK923295.ACS Med. Chem. Lett. 2010; 1: 30-34Crossref PubMed Scopus (36) Google Scholar) (Figure S3D). Also, ΔCENPE cells were ∼2.5-fold more sensitive to Cpd-5, an inhibitor of the essential SAC-kinase MPS1, confirming the synthetic lethal interaction identified in our screen (Figure S3E). ΔCENPE cells displayed a delay in prometaphase, with frequently observed polar chromosomes, typical for loss of CENPE function (Figures S3F and S3G) (Kapoor et al., 2006Kapoor T.M. Lampson M.A. Hergert P. Cameron L. Cimini D. Salmon E.D. McEwen B.F. Khodjakov A. Chromosomes can congress to the metaphase plate before biorientation.Science. 2006; 311: 388-391Crossref PubMed Scopus (322) Google Scholar). These uncongressed chromosomes provoke activation of the SAC, as the mitotic delay was completely prevented by small interfering RNA (siRNA)-mediated depletion of MAD2 (Figure S3F). Consequently, ΔCENPE cells display massive chromosome segregation errors upon SAC perturbation (Figure S3H), explaining the synthetic lethal interaction between loss of the SAC and loss of CENPE. We were surprised to find BUB1, but not BUBR1, as a hit in our screen. BUB1, as well as its binding partner, BUBR1, were previously described to have dual functions in both the SAC and in chromosome alignment (Elowe, 2011Elowe S. Bub1 and BubR1: at the interface between chromosome attachment and the spindle checkpoint.Mol. Cell. Biol. 2011; 31: 3085-3093Crossref PubMed Scopus (85) Google Scholar). Based on this, we anticipated that deletion of either gene would be detrimental to cell viability. Remarkably, BUB1 and BUBR1 were found to be only minimally required for cell fitness of wild-type (WT) HAP1 cells (Figure 3A). Even more surprisingly, loss of BUB1 was synthetic lethal with loss of MAD1 or MAD2, whereas loss of BUBR1 was not (Figure 3A). This indicates that BUB1 and BUBR1 cover separate functions in mitosis. To unravel the molecular details of their functions, we first generated ΔBUB1 HAP1 cells (Figures S1F, 3B, and 3C). As expected, BUBR1 no longer localized to kinetochores in ΔBUB1 cells (Figure 3D) (Johnson et al., 2004Johnson V.L. Scott M.I. Holt S.V. Hussein D. Taylor S.S. Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression.J. Cell Sci. 2004; 117: 1577-1589Crossref PubMed Scopus (264) Google Scholar, Overlack et al., 2015Overlack K. Primorac I. Vleugel M. Krenn V. Maffini S. Hoffmann I. Kops G.J.P.L. Musacchio A. A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint.eLife. 2015; 4: e05269Crossref PubMed Scopus (90) Google Scholar). Consistent with the identification of BUB1 as a hit in our screen, ΔBUB1 cells displayed increased sensitivity to MPS1 inhibition (Figure 3E). This synthetic lethal interaction suggests that ΔBUB1 cells have a defect in chromosome alignment but are able to activate the SAC long enough to allow for full chromosome alignment. Accordingly, ΔBUB1 cells displayed a significant delay in the time to obtain full chromosome alignment as compared to WT cells (Figures 3F and S4A). Also, as predicted, this delay was fully dependent on the SAC, and SAC deficiency led to an enormous increase in chromosome missegregations in ΔBUB1 cells (Figures 3G and 3H). Interestingly, when challenged with nocodazole to trigger a full SAC response, ΔBUB1 cells and WT cells displayed a similar mitotic delay (Figure 3I). This result was somewhat unexpected, since BUB1 has been shown to be essential for the SAC in yeast (Hoyt et al., 1991Hoyt M.A. Totis L. Roberts B.T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function.Cell. 1991; 66: 507-517Abstract Full Text PDF PubMed Scopus (899) Google Scholar), and numerous studies in mammalian cells have shown a SAC defect upon loss of BUB1 (Di Fiore et al., 2015Di Fiore B. Davey N.E. Hagting A. Izawa D. Mansfeld J. Gibson T.J. Pines J. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators.Dev. Cell. 2015; 32: 358-372Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Klebig et al., 2009Klebig C. Korinth D. Meraldi P. Bub1 regulates chromosome segregation in a kinetochore-independent manner.J. Cell Biol. 2009; 185: 841-858Crossref PubMed Scopus (142) Google Scholar, Meraldi and Sorger, 2005Meraldi P. Sorger P.K. A dual role for Bub1 in the spindle checkpoint and chromosome congression.EMBO J. 2005; 24: 1621-1633Crossref PubMed Scopus (176) Google Scholar). To test whether BUB1 possibly fulfills a more subtle, nonessential role in the SAC, we sensitized the ΔBUB1 cells by treatment with a low dose of an MPS1 inhibitor (Santaguida et al., 2010Santaguida S. Tighe A. D’Alise A.M. Taylor S.S. Musacchio A. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.J. Cell Biol. 2010; 190: 73-87Crossref PubMed Scopus (350) Google Scholar). This treatment significantly shortened the duration of the SAC-dependent mitotic delay specifically in ΔBUB1 cells (Figure 3J). This is in line with several other studies that did not observe a role for BUB1 in the SAC or only after sensitization with checkpoint kinase inhibitors (Johnson et al., 2004Johnson V.L. Scott M.I. Holt S.V. Hussein D. Taylor S.S. Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression.J. Cell Sci. 2004; 117: 1577-1589Crossref PubMed Scopus (264) Google Scholar, Vleugel et al., 2015Vleugel M. Hoek T.A. Tromer E. Sliedrecht T. Groenewold V. Omerzu M. Kops G.J.P.L. Dissecting the roles of human BUB1 in the spindle assembly checkpoint.J. Cell Sci. 2015; 128: 2975-2982Crossref PubMed Scopus (53) Google Scholar, Zhang et al., 2015Zhang G. Lischetti T. Hayward D.G. Nilsson J. Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint.Nat. Commun. 2015; 6: 7162Crossref PubMed Scopus (67) Google Scholar). However, since these latter studies all relied on RNA-mediated repression of BUB1, they could not rule out that partial loss of checkpoint function was due to incomplete knockdown. Our results show that complete loss of BUB1 results in a minor checkpoint defect that can only be observed if MPS1 function is compromised. We next wanted to resolve how BUB1 contributes to SAC activity. BUB1 is known to phosphorylate H2A on Thr120, which controls Aurora B recruitment to the inner centromere, establishing a positive feedback loop between Aurora B and MPS1. Interestingly, a GFP-fused kinase-dead version of BUB1 (D946N) was able to rescue the SAC defect to the same extent as a WT BUB1 construct (Figure 3K), without restoring H2A pT120 and Aurora B recruitment (Figures 4D and 4E ), suggesting that the Aurora B localization defect does not contribute to the observed SAC defect. Alternatively, BUB1 could contribute to MCC assembly by recruiting individual MCC components to kinetochores. Indeed, a BUB1 construct lacking a conserved domain (BUB1 Δ437–521) involved in the recruitment of MAD1 (Ji et al., 2017Ji Z. Gao H. Jia L. Li B. Yu H. A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling.eLife. 2017; 6: 431Crossref Scopus (91) Google Scholar, Zhang et al., 2017Zhang G. Kruse T. López-Méndez B. Sylvestersen K.B. Garvanska D.H. Schopper S. Nielsen M.L. Nilsson J. Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling.Nat. Commun. 2017; 8: 15822Crossref PubMed Scopus (58) Google Scholar) and the RZZ complex (Zhang et al., 2015Zhang G. Lischetti T. Hayward D.G. Nilsson J. Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint.Nat. Commun. 2015; 6: 7162Crossref PubMed Scopus (67) Google Scholar) and a construct lacking the ABBA motif (BUB1 1–500), responsible for recruiting CDC20 to kinetochores (Di Fiore et al., 2015Di Fiore B. Davey N.E. Hagting A. Izawa D. Mansfeld J. Gibson T.J. Pines J. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators.Dev. Cell. 2015; 32: 358-372Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Vleugel et al., 2015Vleugel M. Hoek T.A. Tromer E. Sliedrecht T. Groenewold V. Omerzu M. Kops G.J.P.L. Dissecting the roles of human BUB1 in the spindle assembly checkpoint.J. Cell Sci. 2015; 128: 2975-2982Crossref PubMed Scopus (53) Google Scholar), were unable to rescue SAC activity (Figure 3K), consistent with previous results (Vleugel et al., 2015Vleugel M. Hoek T.A. Tromer E. Sliedrecht T. Groenewold V. Omerzu M. Kops G.J.P.L. Dissecting the roles of human BUB1 in the spindle assembly checkpoint.J. Cell Sci. 2015; 128: 2975-2982Crossref PubMed Scopus (53) Google Scholar, Zhang et al., 2015Zhang G. Lischetti T. Hayward D.G. Nilsson J. Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint.Nat. Commun. 2015; 6: 7162Crossref PubMed Scopus (67) Google Scholar). Taken together, our data indicate that the minor role of BUB1 in the SAC is mediated via its interaction with CDC20 as well as through its conserved domain implicated in MAD1 and RZZ-complex recruitment. Next, we set out to determine which domain of BUB1 is responsible for its chromosome alignment function. Because the timing defect of chromosome congression is relatively mild in the haploid ΔBUB1 cells (Figure 3F), we studied alignment in tetraploid ΔBUB1 clones (Figure S4B). We first focused on Aurora B, as Aurora B has important functions in error correction (Horst and Lens, 2014Horst A. Lens S.M.A. Cell division: control of the chromosomal passenger complex in time and space.Chromosoma. 2014; 123: 25-42Crossref PubMed Scopus (91) Google Scholar) and its centromere localization depends on BUB1. As expected, phosphorylation of H2A on Thr120 was completely abrogated in the ΔBUB1 cells (Figures 4A and 4B), and this coincided with a more dispersed localization of Aurora B at the centromere (Figure 4C; Kawashima et al., 2010Kawashima S.A. Yamagishi Y. Honda T. Ishiguro K. Watanabe Y. Phosphorylation of H2A by Bub1 prevents chromosomal instability through localizing shugoshin.Science. 2010; 327: 172-177Crossref PubMed Scopus (349) Google Scholar). Strikingly, despite the fact that the kinase-dead BUB1 mutant failed to restore H2A phosphorylation and Aurora B recruitment to the centromeres (Figures 4D and 4E), both WT and the kinase-dead BUB1 mutant were able to fully rescue chromosome alignment (Figure 4F). Thus, the defect in Aurora B recruitment to centromeres cannot be the cause of the congression defects observed in ΔBUB1 cells. BUB1 is involved in the recruitment of the RZZ complex (composed of ROD, ZW10, and Zwilch) to kinetochores (Caldas et al., 2015Caldas G.V. Lynch T.R. Anderson R. Afreen S. Varma D. DeLuca J.G. The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells.Open Biol. 2015; 5 (150160–150160)Crossref PubMed Scopus (36) Google Scholar, Zhang et al., 2015Zhang G. Lischetti T. Hayward D.G. Nilsson J. Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint.Nat. Commun. 2015; 6: 7162Crossref PubMed Scopus (67) Google Scholar), and this complex was previously shown to contribute to chromosome congression. Consistent with the possibility that BUB1 regulates chromosome congression via the RZZ complex, we found that ROD was a prominent hit in our screen, displaying similar behavior as BUB1 (Figures 5A and 3A). Loss of ZW10 and KNL-1 (which is upstream of the RZZ complex) was detrimental in all of the cell lines, including the parental HAP1 (Figure 5A), implying that both proteins have additional functions independent of the RZZ complex, in line with other reports (Ghongane et al., 2014Ghongane P. Kapanidou M. Asghar A. Elowe S. Bolanos-Garcia V.M. The dynamic protein Knl1 - a kinetochore rendezvous.J. Cell Sci. 2014; 127: 3415-3423Crossref PubMed Scopus (28) Google Scholar, Varma et al., 2006Varma D. Dujardin D.L. Stehman S.A. Vallee R.B. Role of the kinetochore/cell cycle checkpoint protein ZW10 in interphase cytoplasmic dynein function.J. Cell Biol. 2006; 172: 655-662Crossref PubMed Scopus (54) Google Scholar). Deletion of Zwilch, on the other hand, had no effect on cell viability, even in SAC-deficient cells, implying that ROD and ZW10 have additional functions that are not shared by Zwilch and consistent with the finding that ROD and ZW10 can target to kinetochores independently of Zwilch (Gama et al., 2017Gama J.B. Pereira C. Simões P.A. Celestino R. Reis R.M. Barbosa D.J. Pires H.R. Carvalho C. Amorim J. Carvalho A.X. et al.Molecular mechanism of dynein recruitment to kinetochores by the Rod–Zw10–Zwilch complex and Spindly.J. Cell Biol. 2017; 216: 943-960Crossref PubMed Scopus (71) Google Scholar). The fact that ROD is a hit in our screen implies that the RZZ complex is important for chromosome congression but cannot be essential for SAC function, as was suggested previously (Chan et al., 2000Chan G.K. Jablonski S.A. Starr D.A. Goldberg M.L. Yen T.J. Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores.Nat. Cell Biol. 2000; 2: 944-947Crossref PubMed Scopus (166) Google Scholar, Kops et al., 2005Kops G.J. Kim Y. Weaver B.A. Mao Y. McLeod I. Yates 3rd, J.R. Tagaya M. Cleveland D.W. ZW10 links mitotic checkpoint signaling to the structural kinetochore.J. Cell Biol. 2005; 169: 49-60Crossref PubMed Scopus (191) Google Scholar). We therefore generated ΔROD HAP1 cells (Figure S1E) and confirmed that loss of ROD (Figure 5D) disrupted the RZZ complex, since both ZW10 and Spindly no longer localized to kinetochores in ΔROD cells (Figures 5B and 5C) (Scaërou et al., 2001Scaërou F. Starr D.A. Piano F. Papoulas O. Karess R.E. Goldberg M.L. The ZW10 and Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore.J. Cell Sci. 2001; 114: 3103-3114PubMed Google Scholar). In line with our screen data, ΔROD cells displayed an increased sensitivity to MPS1 inhibition and a prominent delay in chromosome congression that was fully dependent on the SAC (Figures 5E–5G). Also, MAD2 depletion in ΔROD cells resulted in a major increase in chromosome missegregations (Figure 5H). Importantly, when ΔROD cells were challenged with nocodazole, they did arrest in mitosis, but the duration of the mitotic arrest was clearly decreased as compared to the parental HAP1 (Figure 5I). This SAC defect is most likely due to the involvement of the RZZ complex in the recruitment of MAD1/2 to kinetochores (Figure 5J) (Caldas et al., 2015Caldas G.V. Lynch T.R. Anderson R. Afreen S. Varma D. DeLuca J.G. The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells.Open Biol. 2015; 5 (150160–150160)Crossref PubMed Scopus (36) Google Scholar, Kops et al., 2005Kops G.J. Kim Y. Weaver B.A. Mao Y. McLeod I. Yates 3rd, J.R. Tagaya M. Cleveland D.W. ZW10 links mitotic checkpoint signaling to the structural kinetochore.J. Cell Biol. 2005; 169: 49-60Crossref PubMed Scopus (191) Google Scholar, Silió et al., 2015Silió V. McAinsh A.D. Millar J.B. KNL1-Bubs and RZZ Provide Two Separable Pathways for Checkpoint Activation at Human Kinetochores.Dev. Cell. 2015; 35: 600-613Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). These results indicate that ΔROD cells have retained a sufficiently functional SAC to be able to delay anaphase until the congression defects that result from loss of RZZ function can be resolved. Importantly, the contribution of the RZZ complex to the SAC is much more prominent than the contribution of BUB1 to the SAC, indicating that there must be a BUB1-independent pool of the RZZ at kinetochores (Qian et al., 2017Qian J. García-Gimeno M.A. Beullens M. Manzione M.G. Van der Hoeven G. Igual J.C. Heredia M. Sanz P. Gelens L. Bollen M. An attachment-independent biochemical timer of the spindle assembly checkpoint.Mol. Cell. 2017; 68: 715-730Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, Silió et al., 2015Silió V. McAinsh A.D. Millar J.B. KNL1-Bubs and RZZ Provide Two Separable Pathways for Checkpoint Activation at Human Kinetochores.Dev. Cell. 2015; 35: 600-613Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Indeed, when we stained for ZW10 in the ΔBUB1 cells, we did not find a reduction in ZW10 but did observe a slight increase in ZW10 levels when BUB1 was overexpressed (Figures 5K, S5A, and S5B), which was completely dependent on its previously identified RZZ-binding domain (BUB1 Δ437–521) (Zhang et al., 2015Zhang G. Lischetti T. Hayward D.G. Nilsson J. Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint.Nat. Commun. 2015; 6: 7162Crossref PubMed Scopus (67) Google Scholar). These data suggest that although BUB1 does have the capacity to recruit the RZZ complex, it is not the main anchor for the RZZ complex at kinetochores. As a final confirmation, we tested whether BUB1 binding to the RZZ complex is important for its alignment function by performing a rescue experiment using the BUB1 RZZ-binding mutant. We found that this mutant was able to fully rescue chromosome alignment (Figure 5L), excluding the RZZ complex as a factor through which BUB1 regulates alignment. Since we excluded BUB1 as the core recruitment factor of the RZZ complex, we investigated the possibility of Zwint-1 fulfilling this function (Kasuboski et al., 2011Kasuboski J.M. Bader J.R. Vaughan P.S. Tauhata S.B.F. Winding M. Morrissey M.A. Joyce M.V. Boggess W. Vos L. Chan G.K. et al.Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores.Mol. Biol. Cell. 2011; 22: 3318-3330Crossref PubMed Scopus (42) Google Scholar, Kops et al., 2005Kops G.J. Kim Y. Weaver B.A. Mao Y. McLeod I. Yates 3rd, J.R. Tagaya M. Cleveland D.W. ZW10 links mitotic checkpoint signaling to the structural kinetochore.J. Cell Biol. 2005; 169: 49-60Crossref PubMed Scopus (191) Google Scholar, Wang et al., 2004Wang H. Hu X. Ding X. Dou Z. Yang Z. Shaw A.W. Teng M. Cleveland D.W. Goldberg M.L. Niu L. Yao X. Human Zwint-1 specifies localization of Zeste White 10 to kinetochores and is essential for mitotic checkpoint signaling.J. Biol. Chem. 2004; 279: 54590-54598Crossref PubMed Scopus (99) Google Scholar). Indeed, our screen revealed that loss of Zwint-1 was synthetic lethal with loss of the SAC, similar to ROD (Figure S5C). We next generated a HAP1 knockout clone for Zwint-1" @default.
• W2787103295 created "2018-02-23" @default.
• W2787103295 creator A5011393666 @default.
• W2787103295 creator A5022409300 @default.
• W2787103295 creator A5022915602 @default.
• W2787103295 creator A5062158667 @default.
• W2787103295 creator A5071251391 @default.
• W2787103295 creator A5074565662 @default.
• W2787103295 creator A5082114523 @default.
• W2787103295 date "2018-02-01" @default.
• W2787103295 modified "2023-10-17" @default.
• W2787103295 title "BUB1 Is Essential for the Viability of Human Cells in which the Spindle Assembly Checkpoint Is Compromised" @default.
• W2787103295 cites W1488719611 @default.
• W2787103295 cites W1540858422 @default.
• W2787103295 cites W1963750681 @default.
• W2787103295 cites W1965143113 @default.
• W2787103295 cites W1992756342 @default.
• W2787103295 cites W1997528931 @default.
• W2787103295 cites W2007074379 @default.
• W2787103295 cites W2007877171 @default.
• W2787103295 cites W2018235941 @default.
• W2787103295 cites W2022017714 @default.
• W2787103295 cites W2024178163 @default.
• W2787103295 cites W2028406725 @default.
• W2787103295 cites W2032967041 @default.
• W2787103295 cites W2039517468 @default.
• W2787103295 cites W2048635498 @default.
• W2787103295 cites W2071156666 @default.
• W2787103295 cites W2079321797 @default.
• W2787103295 cites W2084314276 @default.
• W2787103295 cites W2089806746 @default.
• W2787103295 cites W2090578629 @default.
• W2787103295 cites W2096610880 @default.
• W2787103295 cites W2102653975 @default.
• W2787103295 cites W2108677552 @default.
• W2787103295 cites W2118562565 @default.
• W2787103295 cites W2131558408 @default.
• W2787103295 cites W2133159857 @default.
• W2787103295 cites W2139682891 @default.
• W2787103295 cites W2140493627 @default.
• W2787103295 cites W2143471239 @default.
• W2787103295 cites W2146322167 @default.
• W2787103295 cites W2149055925 @default.
• W2787103295 cites W2152603947 @default.
• W2787103295 cites W2153198916 @default.
• W2787103295 cites W2153608256 @default.
• W2787103295 cites W2155607802 @default.
• W2787103295 cites W2155915788 @default.
• W2787103295 cites W2161791847 @default.
• W2787103295 cites W2161965142 @default.
• W2787103295 cites W2167100671 @default.
• W2787103295 cites W2170141910 @default.
• W2787103295 cites W2170709814 @default.
• W2787103295 cites W2171175279 @default.
• W2787103295 cites W2171582828 @default.
• W2787103295 cites W2174686320 @default.
• W2787103295 cites W2196302162 @default.
• W2787103295 cites W2217038394 @default.
• W2787103295 cites W2253385540 @default.
• W2787103295 cites W2274715327 @default.
• W2787103295 cites W2277104593 @default.
• W2787103295 cites W2287979113 @default.
• W2787103295 cites W2296760199 @default.
• W2787103295 cites W2343964752 @default.
• W2787103295 cites W2571277506 @default.
• W2787103295 cites W2588526630 @default.
• W2787103295 cites W2598957148 @default.
• W2787103295 cites W2602823966 @default.
• W2787103295 cites W2623243466 @default.
• W2787103295 cites W2767253349 @default.
• W2787103295 doi "https://doi.org/10.1016/j.celrep.2018.01.034" @default.
• W2787103295 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29425499" @default.
• W2787103295 hasPublicationYear "2018" @default.
• W2787103295 type Work @default.
• W2787103295 sameAs 2787103295 @default.
• W2787103295 citedByCount "77" @default.
• W2787103295 countsByYear W27871032952018 @default.
• W2787103295 countsByYear W27871032952019 @default.
• W2787103295 countsByYear W27871032952020 @default.
• W2787103295 countsByYear W27871032952021 @default.
• W2787103295 countsByYear W27871032952022 @default.
• W2787103295 countsByYear W27871032952023 @default.
• W2787103295 crossrefType "journal-article" @default.
• W2787103295 hasAuthorship W2787103295A5011393666 @default.
• W2787103295 hasAuthorship W2787103295A5022409300 @default.
• W2787103295 hasAuthorship W2787103295A5022915602 @default.
• W2787103295 hasAuthorship W2787103295A5062158667 @default.
• W2787103295 hasAuthorship W2787103295A5071251391 @default.
• W2787103295 hasAuthorship W2787103295A5074565662 @default.
• W2787103295 hasAuthorship W2787103295A5082114523 @default.
• W2787103295 hasBestOaLocation W27871032951 @default.
• W2787103295 hasConcept C104317684 @default.
• W2787103295 hasConcept C105696609 @default.
• W2787103295 hasConcept C1491633281 @default.
• W2787103295 hasConcept C175732170 @default.
• W2787103295 hasConcept C185592680 @default.
• W2787103295 hasConcept C192838845 @default.
• W2787103295 hasConcept C2780391303 @default.

• next