Cyclin B1 scaffolds MAD 1 at the kinetochore corona to activate the mitotic checkpoint
2020; Springer Nature; Volume: 39; Issue: 12 Linguagem: Inglês
10.15252/embj.2019103180
ISSN1460-2075
AutoresLindsey Allan, Magda Camacho Reis, Giuseppe Ciossani, Pim J. Huis in ’t Veld, Sabine Wohlgemuth, Geert J.P.L. Kops, Andrea Musacchio, Adrian T. Saurin,
Tópico(s)Cancer-related Molecular Pathways
ResumoArticle23 March 2020Open Access Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint Lindsey A Allan Lindsey A Allan Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK Search for more papers by this author Magda Camacho Reis Magda Camacho Reis Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK Search for more papers by this author Giuseppe Ciossani Giuseppe Ciossani Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Pim J Huis in 't Veld Pim J Huis in 't Veld orcid.org/0000-0003-0234-6390 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Sabine Wohlgemuth Sabine Wohlgemuth Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Geert JPL Kops Geert JPL Kops orcid.org/0000-0003-3555-5295 Oncode Institute, Hubrecht Institute—KNAW and University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Andrea Musacchio Andrea Musacchio orcid.org/0000-0003-2362-8784 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Adrian T Saurin Corresponding Author Adrian T Saurin [email protected] orcid.org/0000-0001-9317-2255 Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK Search for more papers by this author Lindsey A Allan Lindsey A Allan Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK Search for more papers by this author Magda Camacho Reis Magda Camacho Reis Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK Search for more papers by this author Giuseppe Ciossani Giuseppe Ciossani Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Pim J Huis in 't Veld Pim J Huis in 't Veld orcid.org/0000-0003-0234-6390 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Sabine Wohlgemuth Sabine Wohlgemuth Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Geert JPL Kops Geert JPL Kops orcid.org/0000-0003-3555-5295 Oncode Institute, Hubrecht Institute—KNAW and University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Andrea Musacchio Andrea Musacchio orcid.org/0000-0003-2362-8784 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Adrian T Saurin Corresponding Author Adrian T Saurin [email protected] orcid.org/0000-0001-9317-2255 Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK Search for more papers by this author Author Information Lindsey A Allan1, Magda Camacho Reis1, Giuseppe Ciossani2, Pim J Huis in 't Veld2, Sabine Wohlgemuth2, Geert JPL Kops3, Andrea Musacchio2 and Adrian T Saurin *,1 1Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK 2Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany 3Oncode Institute, Hubrecht Institute—KNAW and University Medical Centre Utrecht, Utrecht, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (2020)39:e103180https://doi.org/10.15252/embj.2019103180 See also: C Conde & R Gassmann (June 2020) 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 Cyclin B:CDK1 is the master kinase regulator of mitosis. We show here that, in addition to its kinase functions, mammalian Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminal region of MAD1, and point mutations in this sequence abolish MAD1 corona localisation and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona, and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness arises because Cyclin B1:MAD1 localisation loses dependence on MPS1 kinase after the corona has been established, ensuring that corona-localised MAD1 can still be phosphorylated when MPS1 activity is low. Therefore, this study explains how corona-MAD1 generates a robust SAC signal, and it reveals a scaffolding role for the key mitotic kinase, Cyclin B1:CDK1, which ultimately helps to inhibit its own degradation. Synopsis The spindle assembly checkpoint controls anaphase onset and inactivation of the key mitotic kinase, Cyclin B1-CKD1. A surprising new structural role of Cyclin B1 at the kinetochore corona implicates it as a scaffold in this pathway that keeps its own degradation in check. Cyclin B1 binds directly to an acidic patch in the N-terminus of checkpoint protein MAD1. This binding is critical for localizing MAD1 to the kinetochore's corona. Checkpoint kinase MPS1 phosphorylates corona-localized MAD1 on a key C-terminal catalytic residue. The contribution of corona MAD1 to spindle assembly checkpoint signaling allows unattached kinetochores to generate a robust mitotic arrest. Introduction During mitosis, all duplicated chromosomes must attach correctly to microtubules so they can segregate properly when the cell divides. This attachment is mediated via the kinetochore, which is a giant molecular complex assembled on chromosomes at the centromere (Musacchio & Desai, 2017). As well as attaching to microtubules, the kinetochore must also regulate this process to ensure it occurs correctly. One aspect of this regulation involves the activation of the mitotic checkpoint, otherwise known as the spindle assembly checkpoint (SAC), which blocks mitotic exit until all kinetochores have attached to microtubules. The principle of the SAC is that each unattached kinetochore acts as a factory to produce an inhibitor of mitotic exit, known as the mitotic checkpoint complex or MCC (for further molecular details, see Corbett, 2017). The generation of MCC is so efficient that every single kinetochore signalling centre must eventually be extinguished by microtubule attachment to allow the cell to exit mitosis (Rieder et al, 1995; Dick & Gerlich, 2013). This complicated inactivation process, known as SAC silencing, requires the removal of catalysts that are needed at unattached kinetochores to generate the MCC (Etemad & Kops, 2016). Two key catalysts in this regard are MAD1, which drives the first step in MCC assembly, and MPS1, the kinase responsible for recruiting and phosphorylating MAD1 as well as other components needed for MCC assembly. Kinetochore–microtubule attachment extinguishes these activities because microtubules displace MPS1 from its binding site on NDC80 (Hiruma et al, 2015; Ji et al, 2015) and at the same time they provide a highway onto which dynein motors can travel to strip MAD1 away from kinetochores (Howell et al, 2001; Wojcik et al, 2001; Mische et al, 2008; Sivaram et al, 2009). Removal of both MPS1 and MAD1 is essential for SAC silencing because if either one is artificially tethered to kinetochores, then the SAC fails to switch off and mitotic exit is blocked (Jelluma et al, 2010; Maldonado & Kapoor, 2011). One key unexplained aspect of the SAC concerns the kinetochore binding sites for MAD1. MAD1 is recruited to kinetochores via an established KNL1-BUB1 pathway and, in human cells, by an additional pathway involving the ROD/ZW10/Zwilch (RZZ) complex at the kinetochore's corona (a fibrous crescent that forms around kinetochores to aid the capture of microtubules) (Luo et al, 2018). How exactly MAD1 is recruited to the corona and whether this pool of MAD1 can signal to the SAC are unknown. It is crucial to resolve these issues because it is ultimately the RZZ complex that is stripped by dynein to shut down the SAC, implying that this pool of MAD1 is important for MCC generation (Howell et al, 2001; Wojcik et al, 2001; Mische et al, 2008; Sivaram et al, 2009). However, the corona is positioned some distance away from MPS1 and the proposed catalytic centre for MCC generation at the KNL1/MIS12/NDC80 (KMN) network. Therefore, it remains unclear how MAD1 could signal to the SAC from the corona and it is difficult to resolve this issue without knowledge of how MAD1 binds to this region. We show here that the key mitotic kinase complex—Cyclin B1:CDK1—acts as the physical adaptor that links MAD1 to the corona. MAD1 was recently shown to recruit Cyclin B1 to kinetochores (Alfonso-Perez et al, 2019), and although we do see a partial reduction in kinetochore Cyclin B1 when MAD1 interaction is inhibited, the most penetrant phenotype we observe is the complete loss of corona MAD1. This unanticipated scaffolding function of Cyclin B1 is crucial for a robust SAC response, because it allows corona-tethered MAD1 to respond to low level of kinetochore MPS1 activity. This study therefore reveals how the corona pool of MAD1 signals to the SAC and it explains why MPS1 inhibition and dynein-mediated stripping of the corona are both essential for SAC silencing. Results Cyclin B1:MAD1 interaction facilitates Cyclin B1 and MAD1 localisation to unattached kinetochores The Cyclin B:CDK1 kinase complex is a master regulator of mitosis that is activated during G2 phase of the cell cycle to initiate mitotic entry and degraded after chromosome alignment to induce mitotic exit. Analysis of endogenously tagged Cyclin B1-EYFP localisation in RPE1 cells suggested that its localisation was specifically regulated during mitosis. In particular, Cyclin B1-positive foci appeared after nuclear envelope breakdown and disappeared as mitosis progressed (Fig 1A and Movie EV1). Immunofluorescence analysis demonstrated that this localisation pattern reflects specific binding to unattached kinetochores, which is reminiscent of the checkpoint protein MAD1 (Fig 1B and C). In particular, Cyclin B1 depends on MPS1 activity to be established at this location, but thereafter it became largely insensitive to MPS1 inhibition (Fig 1D and E), as also shown previously for MAD1 (Hewitt et al, 2010; Etemad et al, 2019). Please note that in these and all subsequent quantifications, the vertical bars in the graphs represent the 95% confidence intervals, which can be used for statistical inference by eye (see Materials and Methods for full details; Cumming, 2009). To probe for MAD1 and Cyclin B1 association in cells, we recruited LacI-MAD1 to a LacO array on chromosome 1 in U2OS cells (Janicki et al, 2004). This was sufficient to co-recruit Cyclin B1 in a manner that was dependent on a region between amino acids 41–92 of MAD1 (Fig 1F and G). Therefore, these data are consistent with earlier reports that Cyclin B1 localises to unattached kinetochores (Bentley et al, 2007; Chen et al, 2008) in a manner that is dependent on the N-terminus of MAD1 (Alfonso-Perez et al, 2019; Jackman et al, 2020). Figure 1. Cyclin B1:MAD1 interaction helps both proteins to localise to unattached kinetochores A. Endogenous Cyclin B1-YFP localisation during mitosis live in RPE1 cells (still from Movie EV1). B, C. Immunofluorescence images (B) and quantifications (C) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC. Each dot represents a kinetochore, and data are from 40 kinetochore pairs (13 cells, max 5 kinetochore pairs/cell). D, E. Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole-arrested cells (noco) treated with the MPS1 inhibitors, AZ-3146 (5 μM) or reversine (500 nM), either before (D) or after (E) mitotic entry. F. Immunofluorescence images of LacI-MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome 1. G. Live imaging of Cyclin B1-mCherry (CycB1-mCh) in LacO-U2OS cells transfected with LacI-MAD1-FL (full length: aa 1–718) or various LacI-MAD1 truncations (amino acid numbers indicated). H, I. Immunofluorescence images (H) and quantifications (I) of Cyclin B1 and MAD1 kinetochore levels in control (MAD1-WT) or MAD1β HeLa cells (two independent clones: C13 and C24) treated with nocodazole. J, K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and MAD1β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells). L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CENP-C) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H, I). Data information: For all graphs, each dot represents a cell, except panel (C) where dots represent individual kinetochores. The horizontal lines in the graphs indicate the median, and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap, the difference is considered statistically significant at a level of at least P < 0.05 (see Materials and Methods). All graphs display data that are relative to the controls, which are displayed on the left side of each graph and normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. (D, E, I and L) show 30 cells from 3 experiments, and K shows 40 cells from 4 experiments. Scale bars = 5 μM. Download figure Download PowerPoint To determine the function of Cyclin B1 at kinetochores, we attempted to remove it from this location by knocking down endogenous MAD1 and replacing it with a Cyclin B1-binding defective mutant. However, all of the siRNAs tested only mildly reduced MAD1 protein (results not shown). This may be due to the fact that MAD1 is a very stable protein in cells because it takes over a week to fully deplete MAD1 following genetic deletion (see Rodriguez-Bravo et al, 2014). Therefore, to attempt to fully remove Cyclin B1 from kinetochores, we generated a MAD1α knockout cell line that retains only a MAD1β splice variant lacking exon 4 which encodes the Cyclin B1 binding region (hereafter referred to as MAD1β cells; Appendix Fig S1; Sze et al, 2008). Surprisingly, Cyclin B1 was reduced but still present at unattached kinetochores in MAD1β cells (Fig 1H and I). This was not due to residual interaction with MAD1β because doxycycline-inducible knockout of both MAD1α and MAD1β (McKinley & Cheeseman, 2017) completely removed MAD1 from unattached kinetochores but did not further reduce kinetochore Cyclin B1 (Fig 1J and K; note, the data shown are from 10 days of doxycycline treatment which is the minimum time it takes to fully deplete endogenous MAD1 in this system). Therefore, in contrast to a recent report (Alfonso-Perez et al, 2019), these data demonstrate that MAD1 contributes to Cyclin B1 kinetochore localisation, but it is not the only binding partner for Cyclin B1 at kinetochores. At least one other receptor exists that is sufficient to maintain substantial levels of Cyclin B1 on kinetochores in the absence of MAD1. Although inhibiting MAD1-Cyclin B1 interaction did not abolish Cyclin B1 recruitment to kinetochores, it did cause a dramatic effect on MAD1 localisation itself. As discussed earlier, MAD1 localises to the kinetochores via two separate pathways in human cells: the KNL1-BUB1 pathway at the outer kinetochore and the RZZ pathway at the corona. Figure 1H shows that Cyclin B1 and MAD1 both bind to the corona in wild-type cells, which is present as an expanded region outside of CENP-C. However, when their interaction is prevented in MAD1β cells, it is primarily MAD1 that is lost from the corona, as evidenced by a large reduction in its kinetochore volume (Fig 1L). Therefore, this suggested that Cyclin B1 may act as a scaffold to recruit MAD1 to this region. Although MAD1 is well known to bind the corona (Buffin et al, 2005; Kops et al, 2005; Caldas et al, 2015; Silio et al, 2015; Wynne & Funabiki, 2015; Qian et al, 2017; Luo et al, 2018; Pereira et al, 2018; Rodriguez-Rodriguez et al, 2018; Sacristan et al, 2018; Zhang et al, 2019), an interaction with a corona component has never been mapped in vitro. In fact, the only established way to remove MAD1 from the corona is to deplete RZZ subunits, which simply abolishes corona formation altogether (Pereira et al, 2018; Rodriguez-Rodriguez et al, 2018; Sacristan et al, 2018). Therefore, we next sought to explore whether Cyclin B1 might be the receptor that directly recruits MAD1 to the corona. Cyclin B1 directly scaffolds MAD1 at the corona We first assayed for direct MAD1 and Cyclin B1 interaction by obtaining homogeneously purified recombinant full-length MBP-MAD1:MAD2 (MBP stands for maltose-binding protein, an affinity and stabilisation tag) and Cyclin B1:CDK1 complexes and testing their interaction by size-exclusion chromatography (SEC), which separates proteins based on size and shape. When combined stoichiometrically with MBP-MAD1:MAD2, Cyclin B1:CDK1 underwent a prominent shift in elution volume and co-eluted with MAD1:MAD2, indicative of a binding interaction (Fig 2A). Early elution of MAD1:MAD2 from the SEC column reflects its high hydrodynamic radius, typical of highly elongated structures rich in coiled coil. As expected, the elution volume of MBP-MAD1:MAD2 was not affected by the interaction with Cyclin B1:CDK1. Figure 2. Cyclin B1 and MAD1 interact directly through an N-terminal acidic patch on MAD1 Elution profiles and SDS–PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red) and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS–PAGE are also displayed as reference in panels (D and F) to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS–PAGE are also displayed in panels (A–C). Elution profiles and SDS–PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red) and their combination (green). Elution profiles and SDS–PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red) and their combination (green). Elution profiles and SDS–PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1∆93:MAD2 (green) and their combination (red). Elution profiles and SDS–PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD11–92-SNAP (red) and their combination (green). Elution profiles and SDS–PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K and E56K mutations; red) and their combination (green). In (A-D), individual potential binding partners were combined at a concentration of 5 μM. Alignment of the N-terminal region of Cyclin B1 that contains the MAD1-binding region. Numbering refers to the human MAD1 sequence. *conserved, negatively charged residues in MAD1 (E52K, E53K and E56K) required for MAD1:Cyclin B1 interaction. Download figure Download PowerPoint In the absence of Cyclin B1, CDK1 did not interact directly with MAD1:MAD2 (Fig 2B). However, Cyclin B1 on its own did interact with MAD1:MAD2 (Fig 2C). Removal of residues 1–93 from MAD1 (MAD1Δ93:MAD2), which are outside of the predicted coiled-coil domain of MAD1, abolished the interaction with Cyclin B1 (Fig 2D), indicating that residues 1–93 of MAD1 are necessary for the interaction. Importantly, the N-terminal region of MAD1 alone was also sufficient to bind Cyclin B1:CDK1, as revealed by SEC experiments with MBP-MAD11–92-SNAP and Cyclin B1:CDK1 (Fig 2E). Like the full-length MAD1:MAD2 complex, MBP-MAD11–92-SNAP bound to isolated Cyclin B1 but not to CDK1 (Fig EV1A and B). Therefore, MAD1 binds directly to Cyclin B1:CDK1 through a region located in the first 92 residues of MAD1. Click here to expand this figure. Figure EV1. Cyclin B1, not CDK1, interacts with the MAD1:MAD2 complex via the MAD1 N-terminus A–F. Elution profiles and SDS–PAGE for SEC runs on the indicated columns of (A) CDK1 (blue), MBP-MAD11–92-SNAP (red; note that the same elution profile and SDS–PAGE are also displayed as reference in panel (B) and in Fig 2E to improve clarity) and their combination (green); (B) Cyclin B1 (blue), MBP-MAD11–92-SNAP (red) and their combination (green). Asterisks in (A and B) indicate free MBP, a byproduct of the MBP-MAD11–92-SNAP purification; (C) Cyclin B1:CDK1 (blue; note that the same elution profile and SDS–PAGE are also displayed as reference in panel (D) and in Fig 2E to improve clarity), MBP-MAD141–92 (red) and their combination (green); (D) Cyclin B1:CDK1 (blue), MBP-MAD160–92 (red) and their combination (green); (E) Cyclin B1:CDK1 (blue), MBP-MAD141–62 (red; note that the same elution profile and SDS–PAGE are also displayed as reference in panel F to improve clarity) and their combination (green). (F) Cyclin B1 (blue), MBP-MAD141–62 (red) and their combination (green). In all experiments, proteins were combined at 5 μM concentration. Download figure Download PowerPoint To narrow this region down further, we performed additional truncations of MAD11–92, which led to the identification of a minimal Cyclin B1:CDK1 binding site in residues 41–62 of MAD1 (Fig EV1C–F). To identify determinants of the interaction between MAD141–62 and Cyclin B1:CDK1, we extensively mutagenised residues in the MAD141–62 segment and on Cyclin B1. Charge reversals at three conserved negatively charged residues in MAD141–62 (E52K, E53K and E56K) abolished binding to Cyclin B1 (preprint: Allan et al, 2019). When introduced into the full-length MAD1:MAD2 construct, the 3EK mutation was sufficient to severely impair binding to Cyclin B1:CDK1 (Fig 2F). To identify potential binding partners on Cyclin B1 for the MAD1 residues E52, E53 and E56, we mutagenised various clusters of positively charged residues on the surface of Cyclin B1, without however identifying a sufficiently penetrant mutant (preprint: Allan et al, 2019). Collectively, these results indicate that MAD1 and Cyclin B1:CDK1 interact directly, and that the interaction is mediated primarily or exclusively by residues 41–62 of MAD1 and by Cyclin B1. In addition, a conserved acidic patch in this N-terminal region of MAD1 is essential for Cyclin B1 interaction (Fig 2G). To assess the effect of inhibiting Cyclin B1:MAD1 interaction in cells, we generated doxycycline-inducible vsv-tagged MAD1-WT or MAD1-3EK HeLa cells and used these to create MAD1 knockouts via CRISPR/Cas9 (with a gRNA targeting exon 3 to knockout MAD1α and MAD1β; Fig 3A). MAD1 localisation was then assessed in nocodazole-arrested cells, which demonstrated that MAD1-WT and MAD1-3EK displayed a similar localisation pattern in early prometaphase, but only the MAD1-WT was able to localise to the corona when it formed in late prometaphase (Fig 3B). This can be seen in the kinetochore volume analysis which demonstrates that MAD1 and ZW10 kinetochore volumes increase in late prometaphase as the corona forms in MAD1-WT cells (Fig 3C and D). However, in MAD1-3EK cells, although ZW10 expands in late prometaphase, MAD1 volumes actually decrease. This represents a total drop in kinetochore MAD1-3EK levels (Fig 3B and E), which is consistent with the fact that the BUB1-dependent pool of MAD1 is reduced by PP2A as mitosis progresses (Qian et al, 2017). Therefore, a MAD1-3EK mutant, which is unable to bind directly to Cyclin B1, is also unable to localise to the corona in nocodazole-arrested cells. This confirms that Cyclin B1 is the scaffold that recruits MAD1 to this region of the kinetochore in human cells. When the corona pool is removed in MAD1-3EK cells, MAD1 kinetochore recruitment is reduced soon after nuclear envelope breakdown (mirroring the localisation and phosphorylation of its other kinetochore receptor, BUB1) (Nijenhuis et al, 2014; Qian et al, 2017). Note that we also generated YFP-tagged MAD1 cells to visualise its localisation live. However, YFP-MAD1-WT and YFP-MAD1-3EK were both absent from the corona, which suggests that a large N-terminal tag affects MAD1 localisation to this region (Fig EV2). This may be why removing the N-terminus of MAD1 was not reported to affect GFP/mCherry-MAD1 kinetochore localisation in previous studies (Rodriguez-Bravo et al, 2014; Alfonso-Perez et al, 2019) and why removal of the RZZ complex does not affect the kinetochore turnover of venus-MAD1 (Zhang et al, 2019). It is also important to note that the N-terminal vsv-tag on MAD1 is not detected at the corona by immunofluorescence (results not shown), suggesting that this region may be buried in an interaction interface. Figure 3. Cyclin B scaffolds MAD1 at the corona A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days. B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 and 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note that early and late prometaphase was defined based on the level of chromatin condensation. C, D. Relative kinetochore volumes occupied by MAD1 (C) and ZW10 (D) (relative to CENP-C) in MAD1-WT and MAD1-3EK cells in early and late prometaphase. E. Quantification of MAD1 kinetochore intensity from indicated MAD1-WT and 3EK clones treated as in (B). Data information: For all graphs, each dot represents a cell, horizontal lines indicate the median, and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap, the difference is considered statistically significant at a level of at least P < 0.05 (see Materials and Methods). All graphs display data that is relative to HeLa early prometaphase controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5 μM. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. YFP-MAD1-WT and YFP-MAD1-3EK do not localise to the corona Western blot analysis of indicated YFP-MAD1-WT and YFP-MAD1-3EK clones treated with doxycycline. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested YFP-MAD1-WT-C19 and 3EK-C6 cells just after nuclear envelope breakdown (early prometaphase—EPM) or later in mitosis when the chromatin is condensed (late prometaphase—LPM). Scale bars = 5 μM. Quantification of MAD1 kinetochore localisation from cells treated as in (B). In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median, and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap, the difference is considered statistically significant at a level of at least P < 0.05 (see Materials and Methods). The graph displays data that are relative to the early prometaphase controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. Sixty cells from 3 experiments. Download figure Download PowerPoint Corona-localised MAD1 generates a robust SAC response The ability of Cyclin B1 to recruit MAD1 to the corona could allow Cyclin B1 to generate the signal that inhibits its own degradation. However, it is unclear whether corona-localised MAD1 can signal directly to the SAC and, if it can, how this differs from the conventional KNL1-BUB1-MAD1 pathway at the outer kinetochore. One major difference is that Cyclin B1:MAD1 localisation to the corona is insensitive to MPS1 inhibition after mitotic entry (Fig 1E), whereas MPS1 activity is continually required to phosphorylate KNL1 (London et al, 2012; Shepperd et al, 2012; Yamagishi et al, 2012; Vleugel et al, 2015b) and BUB1 (London & Biggins, 2014; Mora-Santos et al, 2016; Faesen et al, 2017; Ji et al, 2017; Qian et al, 2017; Zhang et al, 2017) to recruit MAD1 to the outer kinetochore. To investigate this major difference between the two pathways, we tested the response of MAD1-WT and MAD1-3EK cells to MPS1 inhibition. As expected (Hewitt et al, 2010; Etemad et al, 2019), MAD1 was preserved on kinetochores following MPS1 inhibition with AZ-3146 after mitotic entry in MAD1-WT cells (Fig 4A and B). However, in stark contrast, a MAD1-3EK mutant that cannot bind the corona was completely lost from kinetochores under identical conditions (Fig 4A and B). This sensitivity to MPS1 inhibition was also mirrored by the MAD1-binding partner MAD2, a key downstream component of the MCC (Fig 4A and C). This has considerable impact on the SAC, because MAD1-3EK cells are exquisitely sensitive to MPS1 inhibition in nocodazole, as demonstrated by the fact that these cells immediately exit mitosis at a dose of AZ-3146 that can be tolerated for several hours in MAD1-WT cells (Fig 4D). These data demonstrate that Cyclin B1:MAD1 recruitment becomes insensitive to MPS1 inhibition once the corona has been established, and this subsequently allows the
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