A highly conserved pocket on PP2A‐B56 is required for hSgo1 binding and cohesion protection during mitosis
2021; Springer Nature; Volume: 22; Issue: 7 Linguagem: Inglês
10.15252/embr.202052295
ISSN1469-3178
AutoresYumi Ueki, Michael A. Hadders, Melanie Weisser, Isha Nasa, Paula Sotelo‐Parrilla, Lauren Cressey, Tanmay Gupta, Emil Peter Thrane Hertz, Thomas Kruse, Guillermo Montoya, A. Arockia Jeyaprakash, Arminja N. Kettenbach, Susanne M.A. Lens, Jakob Nilsson,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoReport11 May 2021Open Access Source DataTransparent process A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis Yumi Ueki Yumi Ueki orcid.org/0000-0003-2898-3267 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Michael A Hadders Michael A Hadders orcid.org/0000-0002-2653-2625 Oncode Institute and Center for Molecular Medicine, University Medical Center Utrech, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Melanie B Weisser Melanie B Weisser orcid.org/0000-0003-3946-3929 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Isha Nasa Isha Nasa orcid.org/0000-0001-7699-795X Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Search for more papers by this author Paula Sotelo-Parrilla Paula Sotelo-Parrilla Wellcome Center for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Lauren E Cressey Lauren E Cressey Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Search for more papers by this author Tanmay Gupta Tanmay Gupta orcid.org/0000-0002-4882-0804 Wellcome Center for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Emil P T Hertz Emil P T Hertz The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Thomas Kruse Thomas Kruse orcid.org/0000-0002-2619-7388 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Guillermo Montoya Guillermo Montoya orcid.org/0000-0002-8269-3941 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author A Arockia Jeyaprakash A Arockia Jeyaprakash orcid.org/0000-0002-1889-8635 Wellcome Center for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Arminja Kettenbach Arminja Kettenbach orcid.org/0000-0003-3979-4576 Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Search for more papers by this author Susanne M A Lens Susanne M A Lens orcid.org/0000-0003-2199-7594 Oncode Institute and Center for Molecular Medicine, University Medical Center Utrech, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Jakob Nilsson Corresponding Author Jakob Nilsson [email protected] orcid.org/0000-0003-4100-1125 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Yumi Ueki Yumi Ueki orcid.org/0000-0003-2898-3267 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Michael A Hadders Michael A Hadders orcid.org/0000-0002-2653-2625 Oncode Institute and Center for Molecular Medicine, University Medical Center Utrech, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Melanie B Weisser Melanie B Weisser orcid.org/0000-0003-3946-3929 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Isha Nasa Isha Nasa orcid.org/0000-0001-7699-795X Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Search for more papers by this author Paula Sotelo-Parrilla Paula Sotelo-Parrilla Wellcome Center for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Lauren E Cressey Lauren E Cressey Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Search for more papers by this author Tanmay Gupta Tanmay Gupta orcid.org/0000-0002-4882-0804 Wellcome Center for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Emil P T Hertz Emil P T Hertz The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Thomas Kruse Thomas Kruse orcid.org/0000-0002-2619-7388 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Guillermo Montoya Guillermo Montoya orcid.org/0000-0002-8269-3941 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author A Arockia Jeyaprakash A Arockia Jeyaprakash orcid.org/0000-0002-1889-8635 Wellcome Center for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Arminja Kettenbach Arminja Kettenbach orcid.org/0000-0003-3979-4576 Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Search for more papers by this author Susanne M A Lens Susanne M A Lens orcid.org/0000-0003-2199-7594 Oncode Institute and Center for Molecular Medicine, University Medical Center Utrech, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Jakob Nilsson Corresponding Author Jakob Nilsson [email protected] orcid.org/0000-0003-4100-1125 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Yumi Ueki1, Michael A Hadders2, Melanie B Weisser1, Isha Nasa3, Paula Sotelo-Parrilla4, Lauren E Cressey3, Tanmay Gupta4, Emil P T Hertz1, Thomas Kruse1, Guillermo Montoya1, A Arockia Jeyaprakash4, Arminja Kettenbach3, Susanne M A Lens2 and Jakob Nilsson *,1 1The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark 2Oncode Institute and Center for Molecular Medicine, University Medical Center Utrech, Utrecht University, Utrecht, The Netherlands 3Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, USA 4Wellcome Center for Cell Biology, University of Edinburgh, Edinburgh, UK *Corresponding author. Tel: +45 21328025; E-mail: [email protected] EMBO Reports (2021)22:e52295https://doi.org/10.15252/embr.202052295 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 The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A-B56 phosphatase through a coiled-coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. We find that PP2A-B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis. SYNOPSIS The interaction between hSgo1 and PP2A-B56 is investigated which uncovers a conserved pocket on the B56 subunit required for cohesion protection. PP2A-B56 is shown to negatively regulate hSgo1-cohesin interaction by dephosphorylating hSgo1. A highly conserved pocket on B56 is required for hSgo1 binding and cohesion protection. hSgo1 competes with LxxIxE containing proteins for binding to PP2A-B56. PP2A-B56 dephosphorylates hSgo1 T346 to negatively regulate cohesin binding. Introduction The shugoshin proteins (hSgo1 (Sgol1) and hSgo2 (Sgol2) in humans) are conserved protectors of centromeric cohesion by preventing premature release of the cohesin complex (Marston, 2015). The first shugoshin protein was discovered in Drosophila melanogaster through the isolation of a mutant, MEI-S332, that lost cohesion prematurely during meiosis (Kerrebrock et al, 1992; Kerrebrock et al, 1995; Tang et al, 1998). Subsequent genetic screens identified the shugoshin proteins in yeast (Katis et al, 2004; Kitajima et al, 2004; Marston et al, 2004). Common to these proteins is the presence of an N-terminal coiled coil (CC) region that binds to B56 regulatory subunits, hereby localizing PP2A-B56 to centromeres (Kitajima et al, 2006; Riedel et al, 2006; Tang et al, 2006; Xu et al, 2009). The function of the yeast PP2A-B56-Sgo1 complex during meiosis is to dephosphorylate Rec8, hereby preventing Separase cleavage of cohesin (Brar et al, 2006; Kitajima et al, 2006; Riedel et al, 2006; Ishiguro et al, 2010; Katis et al, 2010). The PP2A-B56 protein phosphatase is a Ser/Thr phosphatase that dephosphorylates numerous substrates to regulate mitosis (Nilsson, 2019; Garvanska & Nilsson, 2020). PP2A-B56 is a trimeric holoenzyme composed of a scaffold subunit (PP2A-A) that connects the B56 subunit with the catalytic subunit (PP2A-C) (Fig 1A) (Xu et al, 2006; Cho & Xu, 2007). The B56 subunit of the holoenzyme confers substrate specificity by binding to interactors that target the phosphatase to its substrates. Most B56 interactors bind via a conserved LxxIxE peptide motif that engages a highly conserved pocket on B56 present in all five B56 isoforms (Hertz et al, 2016; Wang et al, 2016; Wu et al, 2017; Wang et al, 2020). A number of important mitotic regulators such as BubR1, Kif4A, and RacGAP1 bind to PP2A-B56 through a LxxIxE motif to regulate specific dephosphorylation events. There are five isoforms of B56 (B56α, β,γ, δ, and ε) that display distinct localization patterns during mitosis (Foley et al, 2011; Bastos et al, 2014; Vallardi et al, 2019). Figure 1. hSgo1 and LxxIxE motifs compete for binding to PP2A-B56 Structure of the PP2A-B56γ-hSgo1 complex (adapted from Xu et al, PDB: 3FGA). The hSgo1 coiled-coil homodimer interacts with both PP2A catalytic and B56 regulatory subunits. The model shows a LxxIxE peptide bound to B56 at its conserved binding pocket. YFP pull down from cells stably expressing YFP (control) or YFP-B56α enriches the entire PP2A-B56α holoenzyme on the beads. PP2A-A, scaffold subunit; PP2A-C, PP2A catalytic subunit. Coomassie-stained SDS–PAGE of the purified hSgo1 full length (FL) and hSgo11-155. Competition assay with the purified hSgo1 proteins shown in (C). Binding of YFP-B56α to indicated proteins was determined. Representative of 3 independent experiments. Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 (LxxAxA). Binding of YFP-B56α to indicated proteins was determined and quantified by LI-COR. A PP2A-B56γ-BubR1516–715 complex was reconstituted (blue box) and peak fractions pooled and incubated with hSgo11–155. Following incubation, this complex was analyzed by size exclusion chromatography and fractions analyzed by SDS–PAGE (black box). YFP-B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins. Representative of 4 independent experiments. Source data are available online for this figure. Source Data for Figure 1 [embr202052295-sup-0007-SDataFig1.xlsx] Download figure Download PowerPoint In human somatic cells, hSgo1 recruit PP2A-B56α/ε and to a lesser extent the other PP2A-B56 isoforms, to the centromere (Meppelink et al, 2015; Vallardi et al, 2019). This protects cohesin complexes from the mitotic prophase pathway by locally antagonizing mitotic kinase activity and thus WAPL mediated removal of cohesin (Salic et al, 2004; Kitajima et al, 2005; McGuinness et al, 2005). Although hSgo2 has been reported to recruit the bulk of PP2A-B56α to centromeres, hSgo2 is not needed for cohesion protection during mitosis (Kitajima et al, 2006; Tang et al, 2006; Orth et al, 2011; Vallardi et al, 2019). Instead, hSgo2 protects Rec8 from cleavage by separase during meiosis (Lee et al, 2008). In contrast, depleting hSgo1 prevents cohesion protection despite having limited effect on PP2A-B56 centromeric levels (Kitajima et al, 2006; Tang et al, 2006; Vallardi et al, 2019). hSgo1 performs cohesion protection through a conserved cohesin binding motif that is absent from hSgo2 (Liu et al, 2013; Nishiyama et al, 2013; Hara et al, 2014). This hSgo1 cohesin binding motif is phosphorylated by Cdk1 during mitosis on Thr346 to promote cohesin binding (Liu et al, 2013). hSgo1 furthermore competes directly with the cohesin release factor WAPL for cohesin binding to prevent WAPL activity (Hara et al, 2014). Two proteins, Sororin and the cohesin subunit SA2, have been shown to be dephosphorylated by PP2A-B56-hSgo1 to protect cohesin (Hauf et al, 2005; Liu et al, 2013; Nishiyama et al, 2013). Indeed, expressing variants of Sororin and SA2 that cannot be phosphorylated bypass the need for hSgo1 (Liu et al, 2013; Nishiyama et al, 2013). Whether PP2A-B56 bound to hSgo1 dephosphorylates other substrates is unclear. In addition to recruiting PP2A-B56, the shugoshin proteins also recruit the chromosomal passenger complex (CPC) to centromeres through their CC region (Kawashima et al, 2007; Vanoosthuyse et al, 2007; Tsukahara et al, 2010). The shugoshin-dependent localization of CPC to the centromere could also contribute to cohesion protection (Hengeveld et al, 2017). Although the interplay between shugoshin recruitment of PP2A-B56 and the CPC to centromeres is not fully established, recent work suggests that the ability of hSgo1/2 to recruit the CPC and PP2A-B56 are distinct activities (Bonner et al, 2020). Crystallographic studies have determined the human PP2A-B56γ in complex with a fragment of hSgo1 comprising residues 51–96, which represents most, but not the entire N-terminal CC domain (Xu et al, 2009). This hSgo1 fragment displays less affinity to PP2A-B56γ than longer N-terminal fragments of hSgo1, but sufficient affinity to efficiently bind to PP2A-B56γ under crystallization conditions using high protein concentrations. The structure revealed that the hSgo1 fragment forms a dimer which engages several residues of the last C-terminal HEAT repeat of B56γ and makes contacts to the PP2A catalytic subunit (Fig 1A). Although the crystal asymmetric unit shows a 1:1 interaction between hSgo1 peptide strands and PP2A holoenzymes, the hSgo1 peptide strands are arranged into a parallel CC homodimer, where one fragment is related to the other by a twofold crystallographic symmetry axis (depicted as chain A and Asym in Fig 1A). This arrangement allows them to interact symmetrically with PP2A enzymes on both sides. Thus, one PP2A-B56γ holoenzyme displays interactions with residues from both of the two alpha helices forming one hSgo1 CC region in the crystal, which is again consistent with biochemical experiments showing that dimerization of hSgo1 is required for binding to PP2A-B56γ (Tang et al, 1998; Xu et al, 2009). In the PP2A-B56γ-hSgo1 structure, the LxxIxE binding pocket of B56γ is fully exposed and indeed the N-terminal region of hSgo1 does not appear to contain any recognizable LxxIxE motif. These observations raise the possibility that the PP2A-B56-hSgo1 complex can make higher order complexes with LxxIxE containing proteins, which could be important for mitotic cohesion protection. We explored this possibility, which revealed unanticipated aspects of the PP2A-B56-hSgo1 complex important for understanding cohesion protection during mitosis. Results and Discussion hSgo1 and LxxIxE motifs compete for binding to PP2A-B56 We first determined whether hSgo1 can bind to PP2A-B56 in complex with LxxIxE containing proteins. We generated stable inducible HeLa cell lines that express YFP-tagged B56α (stable inducible HeLa cell lines used throughout unless indicated) and arrested cells in prometaphase using nocodazole. Mitotic cells were collected by mitotic shake-off, and YFP-B56α was purified using a YFP affinity resin. This enriches the entire PP2A-B56α holoenzyme on the beads (Fig 1B) and co-purifies LxxIxE containing proteins such as BubR1 and Kif4A (Hertz et al, 2016). We then incubated the purified YFP-B56α with either recombinantly expressed and purified full-length hSgo1 or an N-terminal fragment of hSgo1 spanning residues 1-155 and washed the complexes (Fig 1C and D). As a control, we treated YFP-B56α purifications with buffer instead of hSgo1. Strikingly, both BubR1 and Kif4a bound to PP2A-B56α in the control samples but were efficiently displaced in the presence of hSgo1 (Fig 1D). We performed a similar experiment in the presence of a high-affinity LxxIxE peptide or the control peptide LxxAxA. The LxxIxE peptide efficiently displaced BubR1 and Kif4A as expected but also reduced hSgo1 binding (Fig 1E). These results suggest that hSgo1 might engage the conserved LxxIxE binding pocket of B56α for binding in cells. To test this with purified components, we reconstituted a PP2A-B56γ-BubR1516-715 complex and isolated the complex by size exclusion chromatography. We then incubated this complex with fivefold excess recombinant hSgo11–155 and following incubation characterized the complexes by size exclusion chromatography (Fig 1F). This revealed the formation of a PP2A-B56γ-hSgo11–155 complex devoid of BubR1516–715, fully consistent with the cellular data. To further substantiate these results, we used a panel of B56α mutants that have mutations in the LxxIxE binding pocket and analyzed their ability to bind hSgo1. YFP-B56α variants were purified from prometaphase arrested cells, and hSgo1 and BubR1 binding was analyzed. Interestingly, all B56α mutants unable to bind BubR1 failed to co-purify hSgo1 (Fig 1G). The reason why the LxxIxE peptide does not fully displace hSgo1, in contrast to the B56 mutants, could reflect that the PP2A-B56-hSgo1 complex is very stable once formed. Collectively, these results indicate that LxxIxE motif-containing proteins and hSgo1 compete for a common binding surface on PP2A-B56α. As hSgo1 does not contain any recognizable LxxIxE motif in its CC region, it likely has a binding site that overlaps with the LxxIxE binding pocket of B56 subunits. The LxxIxE binding pocket of PP2A-B56 is required for cohesion protection The involvement of the B56α LxxIxE binding pocket in hSgo1 binding was surprising, given that hSgo1 binds the less conserved C-terminal HEAT repeat of B56γ in the reported structure of the PP2A-B56γ-hSgo1 complex (Fig 2A). To further analyze this, we investigated the B56α R222E LxxIxE pocket mutant in depth for hSgo1 binding and cohesion protection. We compared this to a B56α mutant (B56α 5A), in which all residues at the reported structural interface with hSgo1 were mutated (B56α 5A:Y365A, H377A, Y381A, L384A, M388A) (Fig 2A). First, we compared the binding of PP2A-B56α to hSgo1 and LxxIxE containing mitotic regulators. Consistent with the reported structure of the PP2A-B56γ-hSgo1 complex, we found that YFP-B56α 5A bound less hSgo1 while maintaining its interactions with BubR1 and Kif4A (Fig 2B). In contrast, B56α R222E (mutation in the LxxIxE binding pocket) lost both binding to hSgo1 and LxxIxE containing proteins. In a reciprocal experiment, cells stably expressing FLAG-tagged B56α variants were transfected with YFP-hSgo1, and then, YFP-hSgo1 was affinity-purified from mitotic cells. Again, we observed impaired binding to both B56α R222E and 5A, with the latter mutant retaining more binding to hSgo1 (Fig 2C). A similar result was obtained using YFP-hSgo2 (Fig EV1A). These experiments strengthen the conclusion that the LxxIxE binding pocket of B56 is an important binding determinant for the human shugoshin proteins. Figure 2. hSgo1 binding to the LxxIxE binding pocket of PP2A-B56 is required for cohesion protection Structure of the reported PP2Aγ-B56-hSgo1 binding interface (top) and residues mutated in the B56α 5A mutant are shown (bottom). IP of YFP-B56α from cells stably expressing the B56α WT, R222E, and 5A followed by immunoblotting of indicated proteins. Representative blots are shown (top). hSgo1 signals were normalized to YFP and plotted (bottom). Error bars represent SD (n = 4). Reciprocal IP of (B). YFP-hSgo1 expression construct was transfected into cells stably expressing FLAG-B56α WT, R222E and 5A, followed by YFP IP and immunoblotting of indicated proteins. Representative blots are shown (top). B56α signals were normalized to YFP and plotted (bottom). Error bars represent SD (n = 4). Representative images of chromosome spreads from the indicated conditions. Scale bar, 5 µm. Quantification of (D). The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 4 independent experiments and the mean and SD are indicated. hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere-targeting domain (CB). Representative images of chromosome spreads are shown. CB targets all the rescue constructs (green) to the centromere. Scale bar, 5 µm. Quantification of (F). The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 3 independent experiments and the mean and SD are indicated. Source data are available online for this figure. Source Data for Figure 2 [embr202052295-sup-0008-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Supporting information related to Fig 2 A. YFP IP from cells stably expressing YFP-hSgo2 and transfected with FLAG-B56α constructs. Representative blots are shown. FLAG-B56α signals were normalized to hSgo2 and plotted. Error bars represent SD (n = 4). B. Validation of the B56 RNAi and rescue system. Endogenous B56α was efficiently depleted 48 h after the RNAi treatment. The RNAi-resistant YFP-B56α rescue constructs were expressed approximately at the endogenous level. C, D. Localization of hSgo1(C) and hSgo2 (D) in cells depleted of B56 and expressing the indicated B56α variants. Representative images from 3 independent experiments are shown. Scale bar, 5 µm. E. Experimental protocol of the live cell imaging shown in (F). F. B56 RNAi and rescue with the indicated B56α RNAi-resistant constructs were performed. Time (min) from nuclear envelop breakdown (NEBD) is indicated. Scale bar, 15 µm. G. The time from NEBD to anaphase was measured from 2 independent live cell imaging experiments. Each circle represents an individual cell. Blue circles indicate the cells that were still arrested at the end of filming, and red circles indicate the cells that died during mitosis. The median is indicated with the red horizontal bars. A Mann–Whitney test was used for statistical comparison. Source data are available online for this figure. Download figure Download PowerPoint We next analyzed the ability of the B56α mutants to support cohesion protection. All B56 isoforms were depleted by RNAi and cells were induced to express RNAi-resistant YFP-B56α variants at endogenous levels (Fig EV1B). This in our hands did not affect hSgo1 or hSgo2 localization to centromeres although we note that PP2A has been found to affect hSgo1 localization in another study (Tang et al, 2006) (Fig EV1C and D). Cells were synchronized in prometaphase using nocodazole and chromosome spreads were stained with CREST and DAPI to analyze cohesin integrity. The distance between the two peak intensities of CREST was measured, as premature cohesin removal results in longer distances. Indeed, depleting all B56 subunits increased the distance between centromeres, which was rescued by expressing B56α wild type (WT) (Fig 2D and E). As anticipated from the interaction studies, B56α R222E did not support cohesion protection at all while B56α 5A surprisingly did (Fig 2D and E). To further substantiate this result, we performed live cell imaging of the same conditions. Removing hSgo1 and consequently centromeric cohesin results in prolonged mitotic arrest because of activation of the spindle assembly checkpoint. Similarly, depleting all B56 isoforms resulted in a prolonged arrest which was rescued by YFP-B56α WT and 5A but not the R222E mutant, thus paralleling the chromosome spread results (Fig EV1-EV4). Consistent with our binding experiments (Fig 2B and C), only YFP-B56α WT displayed clear localization to chromosomes as observed by live cell imaging (Fig EV1F and Movies EV1–EV3). We analyzed the YFP-B56α 5A phenotype over a range of expression levels and even low levels of expression rescued the B56 RNAi. These results show that mutating the LxxIxE binding pocket of B56α abolishes cohesion protection while the reported interface for binding the hSgo1 CC appears less critical for this. Click here to expand this figure. Figure EV2. Validation of hSgo1 KD efficiency and the conservation of the hSgo1 coiled-coil region Validation of the hSgo1 antibody and the hSgo1 RNAi by immunoblotting. While the hSgo1 antibody detects unspecific bands in the whole cell lysates (see input), it is specific for hSgo1 after B56 IP, as the treatment with hSgo1 RNAi completely abolishes hSgo1 signal after 48h. Validation of the hSgo1 antibody and the hSgo1 RNAi by immunofluorescence. Representative immunofluorescent images are shown. Scale bar, 5 µm. The conservation of the hSgo1 coiled-coil region. The residues mutated in Sgo1 3A and 4A are indicated. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Supporting information related to Fig 3F and G A–C. Mitotic U2OS LacO cells expressing hSgo1-LacI-GFP variants or LacI-GFP (control) were stained for CPC components, Aurora B (A) and Borealin (not shown). AuroraB (B) and Borealin (C) signal intensity was quantified, normalized to GFP, and plotted. Each circle represents an individual cell, and the mean is indicated. Representative of at least 3 experiments. D–H. Mitotic U2OS LacO cells expressing hSgo11–130-LacI-GFP variants or LacI-GFP (control) were stained for PP2A-C (D) or CPC components, Aurora B (E) and Borealin (not shown). PP2A-C (F), AuroraB (G), and Borealin (H) signal intensity were quantified, normalized to GFP, and plotted. Each circle represents an individual cell, and the mean is indicated. Representative of at least 3 experiments. Data information: Scale bars (in A, D–E), 5 µm. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Validation of TurboID system and hSgo1KD and rescue in various conditions Blot of stable, doxycycline-inducible TurboID-hSgo1 cells treated with doxycycline and/or biotin as indicated, and probed for hSgo1 or Streptavidin. Volcano plot of TurboID-hSgo1 WT cells treated or untreated with biotin. B56 regulatory subunits (2A5A-E) and centromere as well as kinetochore proteins indicated (UniProt name indicated). hSgo1KD and rescue with the indicated hSgo1 RNAi-resistant constructs ± WAPL KD were performed. The time from nuclear envelop breakdown (NEBD) to anaphase was measured from the live cell imaging. Each circle represents an individual cell. Note that the hSgo1 + WAPL RNAi condition is incorporated into Fig 4G for clarity. Experimental protocol of the live cell imaging with hSgo1 complementation with and without partial B56 depletion shown in (F). WB showing the partial KD of all B56 isoforms. hSgo1 ± partial B56 KD and rescue with the indicated hSgo1 RNAi-resistant constructs were performed. The time from nuclear envelop breakdown (NEBD) to anaphase was measured from the live cell imaging. Each circle represents an individual cell. Blue circles indicate the cells that were still arrested at the end of filming, and red circles indicate the cells that died. The median is indicated with the red horizontal bars. Representative of 2 independent experiments. Source data are available online for this figure. Download figure Download PowerPoint To establish that B56α R222E can assemble an active PP2A holoenzyme capable of cohesion protection, we artificially recruited the B56α mutants to the centromere by fusing them to the centromere-targeting domain of Cenp B (CB). We then asked if in the absence of hSgo1, these B56α mutants supported cohesion protection (Fig EV2A and B for hSgo1 depletion). We performed chromosome spreads and measured the distance between CREST peak intensities. All variants of CB-B56α rescued the cohesion defect when hSgo1 was depleted, arguing that they form functional PP2A complexes (Fig 2F and G). Collectively, our analysis of B56α R222E shows t
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