Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex
2019; Springer Nature; Volume: 38; Issue: 7 Linguagem: Inglês
10.15252/embj.2018100977
ISSN1460-2075
AutoresGang Zhang, Thomas Kruse, Clàudia Guasch Boldú, Dimitriya H. Garvanska, Fabian Coscia, Matthias Mann, Marin Barišić, Jakob Nilsson,
Tópico(s)Cellular transport and secretion
ResumoArticle19 February 2019free access Transparent process Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex Gang Zhang Corresponding Author [email protected] orcid.org/0000-0001-7697-7203 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China Qingdao Cancer Institute, Qingdao, Shandong, China Search for more papers by this author Thomas Kruse Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Claudia Guasch Boldú Cell Division Laboratory, Danish Cancer Society Research Center, Copenhagen, Denmark Search for more papers by this author Dimitriya H Garvanska Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Fabian Coscia Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Matthias Mann orcid.org/0000-0003-1292-4799 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Marin Barisic Cell Division Laboratory, Danish Cancer Society Research Center, Copenhagen, Denmark Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jakob Nilsson Corresponding Author [email protected] orcid.org/0000-0003-4100-1125 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Gang Zhang Corresponding Author [email protected] orcid.org/0000-0001-7697-7203 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China Qingdao Cancer Institute, Qingdao, Shandong, China Search for more papers by this author Thomas Kruse Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Claudia Guasch Boldú Cell Division Laboratory, Danish Cancer Society Research Center, Copenhagen, Denmark Search for more papers by this author Dimitriya H Garvanska Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Fabian Coscia Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Matthias Mann orcid.org/0000-0003-1292-4799 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Marin Barisic Cell Division Laboratory, Danish Cancer Society Research Center, Copenhagen, Denmark Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jakob Nilsson Corresponding Author [email protected] orcid.org/0000-0003-4100-1125 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Gang Zhang *,1,2,3, Thomas Kruse1,‡, Claudia Guasch Boldú4,‡, Dimitriya H Garvanska1,‡, Fabian Coscia1,‡, Matthias Mann1, Marin Barisic4,5 and Jakob Nilsson *,1 1Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China 3Qingdao Cancer Institute, Qingdao, Shandong, China 4Cell Division Laboratory, Danish Cancer Society Research Center, Copenhagen, Denmark 5Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark ‡These authors contributed equally to this work *Corresponding author. Tel: 0045 52615354; E-mail: [email protected] *Corresponding author. Tel: 0045 21328025; E-mail: [email protected] EMBO J (2019)38:e100977https://doi.org/10.15252/embj.2018100977 See also: P Meraldi (April 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Kinetochore localized Mad1 is essential for generating a “wait anaphase” signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod-Zw10-Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1-Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1-Bub1 complex. Synopsis Bub1 and the Rod-ZW10-Zwilch (RZZ) complex are both implicated in recruiting the mitotic checkpoint hub Mad1 to mammalian kineotchores. Quantitative analyses of checkpoint activity in human cells shows that RZZ's main function is Mad1 localization, while Bub1 may contribute an additional catalytic role even at substoichiometric levels. Combined CRISPR/Cas9 genome editing and RNAi knockdown is needed for penetrant removal of Bub1 or Rod1. Both Bub1 and the RZZ complex are required for efficient mitotic checkpoint signaling in human cells. Mad1 kinetochore tethering bypasses requirement for Rod. Residual amounts of Bub1 are sufficient for mitotic checkpoint signaling. Introduction Accurate segregation of chromosomes during cell division depends on a functional spindle assembly checkpoint (SAC) that in response to improperly attached kinetochores generates a “wait anaphase” signal (Musacchio, 2011; Lischetti & Nilsson, 2015). The generation of this signal depends on the recruitment of checkpoint proteins to the outer kinetochore, which facilitates the formation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to Cdc20 (Sudakin et al, 2001; Chao et al, 2012). The MCC is a potent inhibitor of the anaphase-promoting complex/cyclosome (APC/C) in complex with its mitotic co-activator Cdc20, and the presence of the MCC therefore delays anaphase onset (Primorac & Musacchio, 2013; Izawa & Pines, 2015; Alfieri et al, 2016; Yamaguchi et al, 2016). How the kinetochore catalyzes MCC production is a major unresolved question in the field. Answering this requires dissection of the molecular mechanisms of checkpoint protein recruitment to the kinetochore and of the interactions between checkpoint proteins. It is clear that phosphorylation of Met-Glu-Leu-Thr (MELT) repeats in the outer kinetochore protein KNL1 by the checkpoint kinase Mps1 generates binding sites for the checkpoint complexes Bub1/Bub3 and BubR1/Bub3 (London et al, 2012; Shepperd et al, 2012; Yamagishi et al, 2012; Primorac et al, 2013; Vleugel et al, 2013, 2015b; Zhang et al, 2014, 2016). Subsequent phosphorylation of Bub1 by Mps1 then facilitates an interaction between Mad1 and Bub1 a mechanism conserved from yeast to man (London & Biggins, 2014; Mora-Santos et al, 2016; Faesen et al, 2017; Ji et al, 2017; Qian et al, 2017; Zhang et al, 2017). Mad1 is in a stable complex with Mad2 and the recruitment of Mad1/Mad2 to kinetochores is essential because this complex catalyzes the first step in MCC formation by loading Mad2 onto Cdc20 (De Antoni et al, 2005; Faesen et al, 2017; Ji et al, 2017). Indeed, the kinetochore levels of the Mad1/Mad2 complex have been shown to correlate with the strength of the checkpoint signal (Collin et al, 2013; Dick & Gerlich, 2013). Bub1 is the only Mad1 kinetochore receptor in yeast, but the situation is more complex in higher eukaryotes as a three-subunit complex composed of Rod, ZW10, and Zwilch (RZZ complex) contributes to Mad1 localization (Basto et al, 2000; Buffin et al, 2005; Kops et al, 2005; Gama et al, 2017; Mosalaganti et al, 2017). The RZZ complex is able to polymerize, hereby generating the outer corona of the kinetochore which recruits checkpoint proteins and proteins regulating microtubule attachment (Pereira et al, 2018; Rodriguez-Rodriguez et al, 2018; Sacristan et al, 2018). Genetic approaches in Drosophila melanogaster and HAP1 cells as well as antibody injection in human cells have revealed that the RZZ complex is required for checkpoint signaling (Basto et al, 2000; Chan et al, 2000; Raaijmakers et al, 2018). However, an accurate quantitative characterization is still missing and the limited effect of Rod removal in non-transformed cells suggests a minor role of the complex in checkpoint signaling (Silió et al, 2015). Furthermore, the RZZ complex could have additional functions in the checkpoint beyond Mad1 recruitment (Grohme et al, 2018). Bub1 was identified as an essential component of the checkpoint in budding yeast and was subsequently shown to be important in multiple model organisms (Hoyt et al, 1991; Roberts et al, 1994; Basto et al, 2000; Vanoosthuyse et al, 2004; Meraldi & Sorger, 2005; Perera et al, 2007; Klebig et al, 2009). However, two recent studies using CRISPR/Cas9-mediated Bub1 gene deletion in HAP1 and RPE1 cells revealed a minor role of Bub1 in checkpoint signaling (Currie et al, 2018; Raaijmakers et al, 2018). These recent studies question a conserved function for Bub1 in the SAC. One reason for the reported discrepancies on the contribution of the RZZ complex and Bub1 to Mad1 localization and checkpoint signaling could be differences between the large number of cell lines and model systems used to study these proteins. Alternatively, since very efficient depletion is needed to uncover the actual contribution of certain checkpoint proteins (Meraldi & Sorger, 2005), a different explanation could be that small variations in checkpoint protein depletion efficiency strongly influence observed phenotypes. The aim of this study was to quantitatively compare penetrant Bub1 and Rod depletions and dissect their functions on the molecular level, hereby clarifying their respective contributions and roles in the checkpoint. Results RZZ-mediated localization of Mad1 is required for an efficient response to unattached kinetochores To investigate the precise role of the RZZ complex in SAC signaling, we first identified a Rod RNAi oligo and established a depletion protocol that efficiently depleted the RZZ component ZW10 to below 5% on kinetochores in HeLa cells and to ≈1% as determined by quantitative Western blot (Figs 1A, D and E, and 2A). We analyzed the strength of the checkpoint by time-lapse microscopy in the presence of nocodazole—a microtubule poison that generates unattached kinetochores. Measuring the time from nuclear envelope breakdown (NEBD) to mitotic exit provides a quantitative readout of checkpoint activity and depletion of Rod clearly decreased the activity of the SAC (control-depleted cells mean arrest time = 790 min, Rod-depleted cells = 220 min; Fig 1B and C). Importantly, co-transfection with an RNAi-resistant Venus-Rod construct fully restored the checkpoint response in Rod-depleted cells confirming that the observed Rod RNAi phenotype was due to depletion of Rod (Fig 1C). Analysis of Venus-Rod localization revealed that it localized to kinetochores immediately after NEBD consistent with a role in the checkpoint (Fig 1B). A Rod construct lacking the N-terminal β-propeller domain required for polymerization of RZZ was less efficient in supporting checkpoint signaling (Gama et al, 2017; Fig 1C). Depletion of Rod in RPE1 and U2OS cells also decreased the duration of the arrest in nocodazole (Fig 1F). Figure 1. Rod is required for Mad1 localization and checkpoint response to unattached kinetochores Schematic of Rod RNAi depletion and synchronization protocol. Representative still images of control, Rod-depleted cells, and Rod-depleted cells supplemented with RNAi-resistant Venus-Rod treated with nocodazole and time in minutes indicated. Scale bar, 5 μm. Time from NEBD to mitotic exit for the indicated conditions with each circle representing a single cell analyzed and mean time indicated by red line and standard error of mean shown. The number of cells counted and mean time indicated above. A Mann–Whitney U-test was used for statistical analysis (ns: non-significant, ****P ≤ 0.0001). A representative result from at least three independent experiments is shown. Immunofluorescence analysis of ZW10 and Mad1 upon depletion of Rod. Scale bar, 5 μm. Quantification of kinetochore intensities of the indicated proteins in control- or Rod-depleted cells with the signal normalized to CREST levels and Bub1 pSpT normalized to Bub1. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least two independent experiments is shown. Time from NEBD to mitotic exit in control-depleted, Rod-depleted, Bub1-depleted, and Rod- and Bub1-co-depleted HeLa, U2OS, and RPE1 cells. Cells were treated with nocodazole. (Mann–Whitney U-test, ns: non-significant, ****P ≤ 0.0001). A representative result from at least three independent experiments is shown. Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells. Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least two independent experiments is shown. Download figure Download PowerPoint Figure 2. Penetrant Rod removal by combing CRISPR/Cas9 and RNAi A. Western blot analysis of whole-cell extract for the indicated conditions. Quantitative analysis of Rod levels using LI-COR technology is indicated below. The asterisk indicates an unspecific band. B. Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions. Kinetochore signal is normalized to CREST signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least two independent experiments shown. C. Representative still images of unperturbed mitosis for parental cells, Rod C cells, and Rod CR cells with time in minutes indicated. CFP-Histone 3 was used here as the chromosome marker. Scale bar, 5 μm. D, E. Time from NEBD to exit of indicated conditions with cells treated with nocodazole or taxol. Red circles represent cells still arrested in mitosis when the filming stopped (Mann–Whitney U-test, ns: non-significant, ****P ≤ 0.0001). Mean (red line) and standard error of mean (black bar) indicated. A representative result from at least three independent experiments is shown. Download figure Download PowerPoint Consistent with the reported role of the RZZ complex in recruitment of Mad1 to kinetochores, analysis by immunofluorescence revealed that Mad1 levels were reduced by 50% in Rod-depleted cells (45 min after releasing from RO3306 into nocodazole) while all other checkpoint proteins analyzed were not decreased (Figs 1D and E, and EV1A). Importantly, the phosphorylation sites in Bub1 required for Bub1-Mad1 complex formation (Ji et al, 2017; Qian et al, 2017; Zhang et al, 2017) were not affected by Rod depletion arguing that the reduction in Mad1 levels upon Rod depletion was not an indirect effect on Bub1 phosphorylation (Figs 1D and E, and EV1A). Depletion of both Rod and Bub1 efficiently removed Mad1 from kinetochores in HeLa cells and resulted in a strong SAC defect in HeLa, U2OS, and RPE1 cells (Fig 1F and G). Click here to expand this figure. Figure EV1. Rod and Bub1 depletions A. Immunofluorescence images of control-depleted cells and Rod-depleted cells with ZW10, Bub1, Bub1 pSpT, BubR1, and Cdc20 kinetochore staining. The quantification is in Fig 1E. Scale bar, 5 μm. B, C. Analysis of protein levels by Western blot analysis of whole-cell extract from HeLa, Rod C, and Bub1 C cells. * in (B) indicates unspecific band recognized by Rod antibody. Download figure Download PowerPoint Given that low levels of Rod might be sufficient to support SAC signaling, we wanted to address SAC strength upon penetrant removal of Rod. To do this, we used CRISPR/Cas9 technology to target Rod exon 2 in HeLa cells. We never obtained HeLa Rod null clones suggesting that the RZZ complex is essential for viability of HeLa cells. However, multiple clones had reduced levels of Rod while all other SAC proteins or APC/C components analyzed were not affected (Figs 2A and EV1B). We will refer to these cell lines as Rod C throughout. During unperturbed mitosis, alignment of chromosomes in the Rod C cell line was delayed shortly compared to the parental HeLa cell line (Fig 2C and Table 1). However, despite the delay in alignment, the cells waited with initiating anaphase until all chromosomes had congressed. When we depleted Rod by RNAi in the Rod C cell line (referred to as Rod CR), we observed more penetrant removal of Rod compared to parental HeLa cells treated with Rod RNAi, while the effect on Mad1 kinetochore levels was subtle (Fig 2A and B). Despite this small difference in Rod levels, the defect in SAC signaling was more penetrant in Rod CR cells compared to Rod RNAi cells (mean time in nocodazole t = 220 min in Rod RNAi, t = 90 min in Rod CR). Importantly, the defect in checkpoint strength could be restored by exogenous expression of Venus-Rod attesting to a specific removal of Rod (Fig 2D). Similarly, in taxol-arrested cells we observed a clear requirement for Rod in checkpoint signaling and Rod C cells clearly arrested less efficiently in taxol consistent with this drug more readily unmasks perturbations to SAC signaling (Collin et al, 2013; Fig 2E). In Rod CR cells, we observed severe defects in chromosome alignment and cells exiting with unaligned chromosomes consistent with a weakened checkpoint response and the reported role of the RZZ complex in chromosome alignment (Starr et al, 1998; Savoian et al, 2000; Fig 2C and Table 1). Table 1. Analysis of mitotic timing and segregation errors during an unperturbed mitosis Condition NEBD-exit (min) Alignment delay (%) Missegregation at exit (%) n HeLa 40 5 5 42 Rod C 50 8 5 52 Rod CR 105 90 55aa Few unaligned chromosomes at exit. 42 Bub1 C 90 56 5 41 Bub1 CR 85 100 100bb Large number of unaligned chromosomes at exit. 31 Rod CR + Bub1 RNAi 35 100 100 14 Bub1 CR + Rod RNAi 30 100 100 17 a Few unaligned chromosomes at exit. b Large number of unaligned chromosomes at exit. In conclusion, penetrant Rod removal reveals that checkpoint signaling strongly depends on the RZZ complex. Bub1 is required for the checkpoint response to unattached kinetochores in different cell lines Based on our results on Rod, we reasoned that the reported differences on the role of Bub1 in the checkpoint might be caused by small variations in protein levels remaining. We therefore decided to investigate this using CRISPR/Cas9 to lower Bub1 levels and hereby sensitizing cells to Bub1 RNAi depletion as we had done for Rod. We targeted exon 2 and obtained multiple Bub1 cell lines (Bub1 C) that based on Western blot analysis appeared to be Bub1 knockout cell lines (Fig EV1C). Surprisingly, these cell lines had a normal checkpoint response in nocodazole but a slightly weakened response to taxol (Fig 3A and B). However, a number of observations argued that the Bub1 C cell lines still expressed residual levels of Bub1 sufficient to support checkpoint signaling. Firstly, when Bub1 C was exposed to several independent Bub1 RNAi oligos (referred to Bub1 CR), we observed a clear impairment of the SAC response in nocodazole that could be rescued by exogenous Bub1 but not Bub1 lacking its Mad1 binding domain (Bub1ΔCD1; Figs 3A and EV2A). Secondly, by immunofluorescence analysis of mitotic Bub1 C cells we observed very weak kinetochore staining with a phospho-specific Bub1 antibody and this staining disappeared upon subsequent Bub1 RNAi depletion (Fig EV2B). Thirdly, mass spectrometry analysis of Bub3 or BubR1 mitotic purifications, which are stable binding partners of Bub1, revealed multiple Bub1 peptides present in Bub1 C, although with reduced intensity compared to parental cells (Figs 3C and D, and EV2C, Table EV1). Importantly, in Bub1 CR we only detected few low intensity peptides revealing further reduction in Bub1 levels (Fig 3C and D, Table EV1). Contamination between samples was excluded by mass spectrometry analysis of purifications run in parallel but only treated with buffer (Table EV1). We estimate that 4% Bub1 are left in the Bub1 C cell lines based on Bub1 peptide intensities in BubR1 purifications. As expected, the level of MCC components in Bub3 and BubR1 purifications was not affected in agreement with an almost normal checkpoint in Bub1 C cells (Figs 3C and EV2C). MCC levels are also normal in Bub1 CR cells as we collected mitotic arrested cells shortly after entry. Collectively, these results argue that very low levels of Bub1 are sufficient for generating a functional checkpoint signal. Given that CRISPR/Cas9-mediated HAP1 and RPE1 Bub1 KO cells have been generated and reported to have an almost fully functional checkpoint response (Currie et al, 2018; Raaijmakers et al, 2018), we investigated if these cells might also have residual Bub1 left accounting for the functional checkpoint. We therefore obtained these cell lines and depleted Bub1 by 4 different RNAi oligos and monitored checkpoint strength in nocodazole (Fig EV2A). Similar to our HeLa Bub1 C cell lines, we observed that Bub1 depletion impaired the SAC response in HAP1 and RPE1 Bub1 KO cells suggesting that residual Bub1 are left in these cell lines and this is why they have a normal checkpoint response. This was further confirmed by mass spectrometry analysis of BubR1 purifications from these cell lines that revealed multiple Bub1 peptides (Fig 3C and D, and Table EV1). Interestingly, in both our HeLa Bub1 C and in the RPE1 and HAP1 Bub1 KO cells we did not detect any peptides in the very N-terminal part of Bub1, which is where the gRNAs are targeting (Fig EV3 and Table EV1). This might suggest that this part of Bub1 is missing in the cell lines. Figure 3. Bub1 is required for SAC signaling from unattached kinetochores A, B. Time from NEBD to exit in indicated conditions with cells treated with nocodazole or taxol. Red circles represent cells still arrested in mitosis when the filming stopped (Mann–Whitney U-test, ns: non-significant, *P ≤ 0.05, ****P ≤ 0.0001). Mean (red line) and standard error of mean (black bar) indicated. A representative result from at least three independent experiments is shown. C. Mass spectrometry analysis of BubR1 IPs from the indicated cell lines. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions. Data from analysis of three technical repeats of BubR1 purifications with standard deviation indicated. D. Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines. In Bub1 CR, we only detected few peptides from three purifications. Standard deviation indicated. E. Representative still images of unperturbed mitosis for parental cells, Bub1 C cells, and Bub1 CR cells with time in minutes indicated. CFP-Histone 3 was used as the chromosome marker. Scale bar, 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Live cell and mass spectrometry analysis of Bub1 knockout cell lines HeLa Bub1C, HAP1 Bub1 KO, and RPE1 Bub1 KO cells were treated with 3 or 4 Bub1 RNAi oligos, and the time from NEBD to exit was measured in the presence of nocodazole. Mean (red line) and standard error of mean (black bar) indicated (Mann–Whitney U-test, ns: non-significant, ***P ≤ 0.0001, ****P ≤ 0.00001). Representative result from two independent experiments is shown. Staining of indicated cell lines with Bub1 pSpT antibody. Scale bar, 5 μm. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions for Venus-Bub3 purifications from indicated cell lines with standard deviation shown. Mass spectrometry analysis was performed on three technical replicates of Venus purification. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Bub1 peptides detected by mass spectrometryThe primary sequence of Bub1 is shown and peptides identified in HeLa and HeLa Bub1 C cells shown from Venus Bub3 purifications. The site targeted by the gRNA and RNAi oligos is indicated. Download figure Download PowerPoint Depletion of Bub1 by RNAi in Bub1 C cells allowed us to determine the contribution of Bub1 to the SAC and its role in chromosome segregation. In both nocodazole- and taxol-arrested cells, the checkpoint was strongly impaired although not fully abrogated which is due to Rod-mediated recruitment of Mad1 (mean time in nocodazole t = 705 min in Bub1 C, t = 110 min in Bub1 CR; mean time in taxol t = 770 min in Bub1 C, t = 60 min in Bub1 CR; Fig 3A and B). In Bub1 C cells, chromosome alignment was delayed, but cells rarely entered anaphase with unaligned chromosomes due to a functional checkpoint (Fig 3E and Table 1). However, the complete removal of Bub1 resulted in massive alignment problems and cells exiting mitosis with many unaligned chromosomes suggesting a near complete loss of checkpoint activity (Fig 3E and Table 1). Although Bub1 is known to be important for chromosome alignment (Meraldi & Sorger, 2005), we are not aware of previous studies reporting such a strong defect upon Bub1 removal. In summary, we conclude that Bub1 is required for the efficient response to unattached kinetochores in human cells and that 2–4% of Bub1 are sufficient for almost normal checkpoint activity. Furthermore, our quantitative comparison of Rod and Bub1 CR phenotypes suggests that Rod and Bub1 activities are integrated, as their combined individual contributions to SAC strength are considerably lower than that of the control situation. Tethering of Mad1 to kinetochores bypasses the requirement for Rod As both Rod and Bub1 are responsible for Mad1 kinetochore localization and checkpoint activation, we wanted to address whether Mad1 kinetochore localization is their only function or whether they have additional roles in the checkpoint. To address this, we analyzed checkpoint signaling in cells where Mad1 was artificially tethered to kinetochores through fusion to KNL1 or Ndc80 and analyzed checkpoint strength in Bub1 CR, Rod CR or by RNAi depletion (Fig 4A and B). While the Ndc80-Mad1 or Mad1-KNL1 fusion completely bypassed the requirement for Rod in SAC signaling, Bub1 was still required. This result suggests that RZZ is mainly required for Mad1 localization while Bub1 has additional checkpoint functions. In agreement with the Mad1-tethering experiments, we observed cells exiting from nocodazole arrest with reduced but relatively stable Venus-tagged Mad1 localized to kinetochores in cells lacking Bub1 (Fig 4C). This was in contrast to the Mad1 localization in cells lacking Rod where we observed continuously reduced levels of Mad1 at kinetochores with the kinetochore signal disappearing after approximately 1 h. Figure 4. Mad1 localization and kinetochore dynamics A, B. Tethering of Mad1 (Mad1 485–718) to Ndc80, KNL1 (Zhang et al, 2017), or Bub1 1–553ΔCD1 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped. Mean (red line) and standard error of mean (black bar) indicated (Mann–Whitney U-test, ns: non-significant, ****P ≤ 0.0001). A representative result from at least three independent experiments is shown. C. Localization of Venus-tagged Mad1 in parental HeLa cells, Bub1 CR cells, and Rod CR cells in the presence of nocodazole. Scale bar, 5 μm. D. Still images from FRAP experiments using Venus-tagged Mad1 in the indicated conditions. Image before bleach is shown and then time following bleach. Arrowheads indicate kinetochore pairs. Scale bar, 5 μm for whol
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