The PP2AB56 Phosphatase Promotes the Association of Cdc20 with APC/C in Mitosis Sun Joo Lee, Veronica Rodriguez-Bravo*, Hyunjung Kim, Sutirtha Datta and Emily A

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RESEARCH ARTICLE The PP2AB56 phosphatase promotes the association of Cdc20 with APC/C in mitosis Sun Joo Lee, Veronica Rodriguez-Bravo*, Hyunjung Kim, Sutirtha Datta and Emily A. Foley‡

ABSTRACT of mitosis and cytokinesis (reviewed in Pines, 2011; Sivakumar and PP2A comprising B56 regulatory subunit isoforms (PP2AB56)isa Gorbsky, 2015). serine/threonine phosphatase essential for mitosis. At the Mitosis-specific phosphorylation controls the assembly of APC/ Cdc20 Cdc20 kinetochore, PP2AB56 both stabilizes microtubule binding and C and modulates the timing of its activation. APC/C is promotes silencing of the spindle assembly checkpoint (SAC) modified at >100 sites, with both stimulatory and inhibitory through its association with the SAC protein BubR1. Cells depleted phosphorylation sites identified (Chung and Chen, 2003; Craney ’ of the B56 regulatory subunits of PP2A are delayed in activation of et al., 2016; D Angiolella et al., 2003; Fujimitsu et al., 2016; Hein Cdc20-containing APC/C (APC/CCdc20), which is an essential step for and Nilsson, 2016; Jia et al., 2016; Kimata et al., 2008; Labit et al., mitotic exit. It has been hypothesized that this delay arises from 2012; Qiao et al., 2016; Schwab et al., 2001; Tang et al., 2004; increased production of the mitotic checkpoint complex (MCC), an Yudkovsky et al., 2000; Zhang et al., 2016). Perhaps the best- APC/CCdc20 inhibitor formed at unattached kinetochores through SAC understood impact of phosphoregulation on APC/C activation is via signaling. In contrast to this prediction, we show that depletion of B56 the spindle assembly checkpoint (SAC), a signal transduction subunits does not increase the amount or stability of the MCC. cascade initiated at unattached kinetochores. This checkpoint Rather, delays in APC/CCdc20 activation in B56-depleted cells depends on multiple kinases, including Mps1, and culminates in Cdc20 correlate with impaired Cdc20 binding to APC/C. Stimulation of the inhibition of APC/C activity (reviewed in Foley and APC/CCdc20 assembly does not require binding between PP2AB56 Kapoor, 2013; London and Biggins, 2014). The SAC inhibits APC/ Cdc20 and BubR1, and thus this contribution of PP2AB56 towards mitotic exit C by catalyzing the association of Cdc20 with the SAC is distinct from its functions at kinetochores. PP2AB56 associates with proteins Mad2 (also known as MAD2L1), BubR1 (also known as APC/C constitutively in a BubR1-independent manner. A mitotic BUB1B) and Bub3, to form the mitotic checkpoint complex (MCC) phosphorylation site on Cdc20, known to be a substrate of PP2AB56, (Sudakin et al., 2001). Within the MCC, Cdc20 is inhibited because modulates APC/CCdc20 assembly. These results elucidate the it cannot recognize degron sequences (Chao et al., 2012). contributions of PP2AB56 towards completion of mitosis. Additionally, the MCC can bind to and inhibit a second molecule of Cdc20 already bound to the APC/C (Alfieri et al., 2016; Izawa KEY WORDS: Mitosis, PP2A, B56, APC/C, Cdc20 and Pines, 2015; Yamaguchi et al., 2016). Serine/threonine phosphatases facilitate SAC inactivation at the kinetochore to INTRODUCTION permit APC/CCdc20 activation (Espert et al., 2014; Meadows et al., Error-free chromosome segregation during mitosis requires that 2011; Nijenhuis et al., 2014; Rosenberg et al., 2011), but less is replicated sister chromatids attach to microtubules at opposite ends known about whether and how phosphatases impact APC/CCdc20 of the mitotic spindle before sister chromatid separation and exit assembly in mitosis. from mitosis. The fidelity of chromosome segregation depends on Recent data indicate that the PP2A serine/threonine phosphatases dynamic phosphorylation, with contributions from multiple serine/ containing a B56 isoform regulatory subunit (hereafter denoted threonine kinases, including Cdk1–cyclin-B, Plk1, Aurora B and PP2AB56) play essential roles in mitotic progression. The PP2AB56 Mps1 (also known as TTK), leading to the generation of thousands holoenzyme is a heterotrimer consisting of a catalytic subunit of mitosis-specific phosphorylation events (Olsen et al., 2010). One (encoded by PPP2CA and PPP2CB) bound to two non-enzymatic critical way in which phosphorylation influences mitosis is by subunits, the scaffold subunit (encoded by PPP2R1A and controlling the activity of the anaphase-promoting complex/ PPP2R1B) and the B56 regulatory subunit. In human cells, there cyclosome (APC/C), an essential E3 ubiquitin ligase. When are five B56 isoforms (α, β, γ, δ and ε; encoded by PPP2R5A, APC/C is bound to its co-activator Cdc20 (the complex is denoted PPP2R5B, PPP2R5C, PPP2R5D and PPP2R5E, respectively), APC/CCdc20 hereafter), it ubiquitylates securin and cyclin B, leading which share a pseudo-HEAT repeat domain (Cho and Xu, 2007; Xu to their proteasomal degradation. The destruction of securin and et al., 2006). The five isoforms can function redundantly in mitosis cyclin B triggers sister chromatid separation and mitotic exit, (Foley et al., 2011). PP2AB56 substrate selectivity is achieved, at respectively, leading to chromosome segregation, and completion least in part, through protein–protein interactions conferred by B56 subunit binding to a short linear motif sequence of LxxIxE (where Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY L, I and E denote leucine, isoleucine and glutamic acid, 10065, USA. respectively, and x indicates any amino acid) (Hertz et al., 2016). *Present address: Pathology Department, Icahn School of Medicine, Mount Sinai, B56 New York, NY 10029, USA. PP2A dephosphorylates substrates of multiple mitotic kinases, including Mps1, Plk1 and Aurora B, and this activity presumably ‡ Author for correspondence ([email protected]) underlies its myriad of mitotic functions, including preservation of H.K., 0000-0003-1318-7153; E.A.F., 0000-0003-4870-558X sister chromatid cohesion at the centromere (Chen et al., 2007; Kitajima et al., 2006; Riedel et al., 2006; Tang et al., 2006),

Received 13 January 2017; Accepted 28 March 2017 stabilization of microtubule binding at the kinetochore (Foley et al., Journal of Cell Science

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2011), silencing of the SAC at the kinetochore (Espert et al., 2014; in multiple ways, including defects in SAC inactivation, APC/ Nijenhuis et al., 2014), recruitment of E2 ubiquitin-conjugating CCdc20-dependent proteolysis of cyclin B and/or dephosphorylation enzymes to APC/CCdc20 (Craney et al., 2016), and assembly of the of Cdk1–cyclin-B substrates. We determined that siB56 cells were central spindle in anaphase (Bastos et al., 2014). not delayed when mitotic exit was triggered by addition of the Cdk1 During mitosis, a critical pool of PP2AB56 is the fraction inhibitor RO-3306 (Vassilev et al., 2006) (Fig. S1C), suggesting associated with the SAC protein BubR1. The direct binding of B56 that siB56 cells are proficient in dephosphorylating Cdk1 substrates. subunits to BubR1 is mediated by an LxxIxE motif present in This finding is consistent with the B55 family of PP2A regulatory BubR1’s kinetochore attachment regulatory domain (KARD) subunits mediating Cdk1–cyclin-B substrate dephosphorylation in (Suijkerbuijk et al., 2012). Deletion or mutation of BubR1’s human cells (Schmitz et al., 2010). Next, we analyzed the rate of KARD prevents PP2AB56 kinetochore targeting and is known to cyclin B1 proteolysis induced by Mps1 inhibition in CCNB1Venus/+ impair mitosis in two ways. First, kinetochore–microtubule RPE1 cells, in which one allele of cyclin B1 is expressed as a fusion attachments are destabilized (Kruse et al., 2013; Suijkerbuijk with the fluorescent Venus protein (Collin et al., 2013). Cyclin B1 et al., 2012; Xu et al., 2013). Second, SAC silencing at the proteolysis was inefficient in siB56 cells (Fig. 1F,G), suggesting a kinetochore is delayed (Espert et al., 2014; Nijenhuis et al., 2014). potential defect in APC/CCdc20 activation. Finally, we used Collectively, these data establish a critical role for the direct binding quantitative immunofluorescence microscopy to compare between BubR1 and PP2AB56 in mitotic progression, and hence, the kinetochore localization of the SAC proteins Mad2 and BubR1. timing of APC/CCdc20 activity. In the presence of nocodazole and the proteasome inhibitor MG132 Here, we examine human tissue culture cells that had been (to prevent mitotic exit), the kinetochore targeting of Mad2 and depleted of B56 subunits during mitosis. Consistent with previous BubR1 were similar in siCtrl and siB56 cells (Fig. 1H,I). Reversine reports (Espert et al., 2014; Nijenhuis et al., 2014), we find that B56- addition reduced the levels of both Mad2 and BubR1 at the depleted cells exhibit delayed APC/CCdc20 activation and have kinetochore, although kinetochores in siB56 cells did retain more increased recruitment of SAC proteins to the kinetochore. Given that Mad2 and BubR1 compared to siCtrl cells (Fig. 1H,I). The latter SAC signaling at the kinetochore is rate-limiting for the inhibition of result is consistent with previous work indicating that PP2AB56 APC/CCdc20 by MCC (Collin et al., 2013; Dick and Gerlich, 2013), promotes BubR1 eviction at the kinetochore after Mps1 inhibition we expected a possible increase in or stabilization of the MCC in (Espert et al., 2014; Nijenhuis et al., 2014). However, it was unclear B56-depleted cells. However, we find no evidence that cells whether changes in the localization of SAC proteins at the depleted of B56 subunits have increased levels or stability of the kinetochore are the only reason that siB56 cells are delayed in MCC. Rather, we show that PP2AB56 is required to stimulate Cdc20 mitotic exit following Mps1 inhibition. binding to APC/C in mitosis. PP2AB56 is associated with APC/C throughout the cell cycle, in a BubR1-independent manner. We PP2AB56 depletion does not alter the amount or stability of show that residue Ser92 on Cdc20, previously shown to be a the mitotic checkpoint complex substrate of PP2AB56 (Craney et al., 2016; Jia et al., 2016), has a role If persistent SAC activation at the kinetochore is the only defect in in modulating APC/CCdc20 assembly. Taken together, these data mitotic exit in siB56 cells, then we would predict an increase in the reveal a role for PP2AB56 in stimulating the assembly of APC/ amount and/or stability of the MCC. We examined this possibility in CCdc20 complexes in mitosis. three ways. First, we used immunoprecipitation (IP) to compare the levels of MCC in siCtrl and siB56 cells. We used an established RESULTS approach (Collin et al., 2013), outlined in Fig. 2A, performing a PP2AB56 promotes mitotic exit Cdc20 IP of whole-cell lysate to compare total MCC amounts To determine the impact of B56 depletion on the kinetics of (MCCTotal), an APC4 (also known as ANAPC4) IP of whole-cell chromosome segregation, we used a validated set of five siRNAs lysate to compare the pool of MCC bound to APC/C (MCCAPC/C) targeting B56α through B56ε (siB56α-ε) (Foley et al., 2011). and, finally, a Cdc20 IP from APC4-depleted supernatant (Cdc204S siB56α-ε-transfected cells (hereafter referred to as siB56α-ε cells) IP, Fig. 2B) to compare the pool of MCC in excess of the APC/C were delayed in mitosis compared to cells treated with non-targeting (MCCFree). In siB56 cells, there was no increase in BubR1 or Mad2 in control siRNA (siCtrl), and this delay was rescued by expression of Cdc20 IPs, indicating that MCCTotal is not increased by B56 depletion RNA interference (RNAi)-resistant B56α (Fig. 1A). As expected (Fig. 2C, compare lanes 5 and 6). APC4 IPs from siB56 cells (Foley et al., 2011), siB56α-ε cells required more time to achieve contained less BubR1, Mad2 and Cdc20 compared to siCtrl cells, metaphase chromosome alignment (Fig. 1B, blue bars). suggesting that there is also no increase in MCCAPC/C (Fig. 2C, Additionally, siB56α-ε cells were delayed between metaphase compare lanes 3 and 4). Finally, Cdc204S IPs from siB56 cells had and anaphase (Fig. 1B, green bars, quantified in Fig. 1C). reduced levels of BubR1 and Mad2 compared to siCtrl cells (Fig. 2C, Consistent with recent reports (Espert et al., 2014; Nijenhuis compare lanes 7 and 8). Thus, by three assessments of the relative et al., 2014), nocodazole-arrested siB56α-ε cells were delayed in amounts of MCC, we observe no increase in MCC in siB56 cells. mitotic exit following addition of reversine, an inhibitor of the However, we did note decreased association between APC/C and kinase Mps1 (Santaguida et al., 2010) (Fig. 1D). This delay was Cdc20 (Fig. 2C, compare lanes 5 and 6), discussed below. rescued by expression of RNAi-resistant B56ε (Fig. S1A,B), and it Next, we compared the stability of MCC after reversine addition. required co-transfection of siRNAs targeting B56α, B56γ, B56δ and siCtrl and siB56 cells were collected by mitotic shake-off and B56ε, but not B56β (Fig. 1D). Because siRNA against B56β was treated with reversine and MG132. HeLa cells were used in this and not essential for inducing a delay in anaphase onset (Fig. 1C), further large-scale IP experiments requiring RNAi. In the absence of further experiments to deplete PP2AB56 were performed with reversine, Mad2 IPs from siB56 cells contained similar amounts of siRNA targeting B56α, B56γ, B56δ and B56ε (hereafter referred to Cdc20 and less BubR1 compared to siCtrl cells, consistent with as siB56), resulting in a partial depletion of each subunit (Fig. 1E). there being no increase in the amount of MCC (Fig. 2D, compare In siB56-transfected cells (hereafter referred to as siB56 cells), lanes 3 and 7). Reversine disassociated Cdc20 and BubR1 from the delay in mitotic exit after Mps1 inhibition (Fig. 1D) could arise Mad2 with similar kinetics in siCtrl and siB56 cells (Fig. 2D, Journal of Cell Science

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Fig. 1. See next page for legend. compare lanes 3-6 vs 7-10). Similarly, in Cdc20 IPs, reversine- that the MCC is not stabilized in siB56 cells. However, we note that induced disassociation of Mad2 from Cdc20 was unchanged by B56 BubR1 remained associated with Cdc20 upon reversine addition in depletion (Fig. 2E, compare lanes 1-4 vs 5-8). These data suggest both siCtrl and siB56 cells (Fig. 2E), possibly reflecting a Journal of Cell Science

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Fig. 1. PP2AB56 promotes the timing of anaphase and mitotic exit. demonstrating that the defect is specifically due to the reduction in (A–C) PP2AB56 depletion delays anaphase onset. (A,B) RPE1 cells PP2AB56 (Fig. 3A, compare lanes 8 and 9, quantified in Fig. 3B). α – expressing RNAi-resistant B56 and/or H2B mCherry were transfected with Similarly, APC3 (also known as CDC27) IPs from siB56 cells siRNA targeting B56α, B56β, B56γ, B56δ and B56ε (siB56α-ε) or control siRNA (siCtrl) and imaged live. (A) The duration of mitosis was measured for contained less Cdc20 and Mad2 (Fig. 3C, compare lanes 4 and 5, individual cells. Plotted is the fraction of cells that entered anaphase as a quantified in Fig. 3D). We noted that the decrease in APC/C–Cdc20 function of time in mitosis. (B) Representative micrographs. Numbers indicate association differs from that in recent observations (Craney et al., time (min) relative to nuclear envelope breakdown (NEBD). The time to 2016), possibly due to differences in RNAi (in this study we metaphase (blue bar) and time from metaphase to anaphase (green bar) is eliminated siRNA against B56β) or the addition of MG132 by – indicated. (C) RPE1 cells expressing H2B mCherry were treated with siCtrl, Craney and colleagues, which stabilizes APC/CCdc20 complexes siB56α-ε, or a mixture of siRNAs against B56α, B56γ, B56δ and B56ε (siB56) and imaged live. The cumulative anaphase onset is plotted. (D) Depletion of (Kelly et al., 2014). Okadaic acid, which inhibits all PP2A PP2AB56 delays mitotic exit. RPE1 cells were treated with siCtrl, siB56α-ε or holoenzymes as well as PP1, PP4 and PP6 serine/threonine mixtures in which an siRNA against a single B56 isoform had been omitted. phosphatases (Swingle et al., 2007), also reduced the association Cells were treated with nocodazole for 2 h and then imaged live before and of Cdc20, BubR1 and Mad2 with APC4 (Fig. 3E, compare lanes 3 after reversine addition. The median time of mitotic exit (line) and exit time of and 4). Thus, we conclude that phosphatase activity stimulates individual cells (open circles) is plotted. Triangles indicate cells that remained Cdc20 association with APC/C in mitosis. in mitosis at the end of the imaging period. (E) siCtrl and siB56 mitotic HeLa We observed that B56 depletion also results in a decrease in lysates were probed by western blotting. Asterisk indicates non-specific band. (F,G) Cyclin B1 proteolysis is delayed in siB56 cells. (F) RPE1 CCNB1Venus/+ Cdc20 levels (Fig. 3A, compare lanes 1 and 3). In lysates that had siCtrl or siB56 cells were treated as described in D and imaged live. Still images been treated with MG132 before lysis, this decrease was not from differential interference contrast (DIC) and fluorescent imaging are observed (Fig. 2D, compare lanes 1 and 2), suggesting that B56 shown. Numbers indicate time (min) relative to addition of reversine. (G) depletion may enhance proteasome-dependent degradation of Plotted is the fluorescence intensity relative to reversine addition. Each line Cdc20 (Reddy et al., 2007). Therefore, to determine if reduced indicates a single cell and the last time point plotted is either mitotic exit or the co-precipitation of Cdc20 with APC/C is simply due to decreased experimental end-point (150 min). (H,I) siB56 cells have increased recruitment of Mad2 and BubR1 when Mps1 is inhibited. RPE1 siCtrl or siB56 cells were levels of Cdc20, we performed Cdc20 IPs from siCtrl and siB56 incubated with nocodazole and MG132, and treated with or without reversine cells. B56 depletion reduced Cdc20 association with APC1 (also before processing for immunofluorescence microscopy. (H) Maximum known as ANAPC1), APC3, APC4, APC7 and APC8 (also known intensity projection images of representative cells used for quantification as CDC23) (Fig. 3F, compare lanes 4 and 5, quantified in Fig. 3G). shown in I. (I) Quantification of kinetochore recruitment. Each circle represents Cdc20 association with BubR1 and Mad2 was also reduced in siB56 the average kinetochore signal of one cell. Line indicates mean; a.u., arbitrary cells. Taken together, these results indicate that PP2AB56 is required units; n.s., not significant (P>0.05), **P<0.005, ***P<0.0005, Student’s two-tailed t-test. Results are representative of three independent experiments. to promote the association between APC/C and Cdc20. Decreased n indicates number of cells analyzed from three experiments (A,C) or single association between APC/C and Cdc20 would be expected to experiment (D). Scale bars: 5 μm. contribute to the delay in mitotic exit in siB56 cells, synergizing with increased phosphorylation of SAC proteins at the kinetochore requirement for proteasome activity in the complete disassembly of (Espert et al., 2014; Nijenhuis et al., 2014) and defects in E2 MCC (Reddy et al., 2007; Stegmeier et al., 2007; Varetti et al., ubiquitin-conjugating enzyme recruitment to APC/CCdc20 (Craney 2011). Thus, we cannot exclude the possibility that the BubR1– et al., 2016). Cdc20 association is altered by B56 depletion. In our third approach, we compared MCC production at the single PP2AB56-dependent stimulation of APC/CCdc20 assembly cell level (Collin et al., 2013). The basis of this assay is that APC/ does not require the KARD in BubR1 CCdc20-dependent ubiquitylation targets cyclin A2 for proteolysis at We reasoned that PP2AB56 binding to the BubR1 KARD could the onset of mitosis. Cyclin A2 and MCC compete for Cdc20 access potentially mediate its role in promoting APC/CCdc20 assembly. To and therefore the rate of cyclin A2 proteolysis is inversely test this possibility, we first examined BubR1-associated proteins in proportional to the level of MCC (Collin et al., 2013; Di Fiore siCtrl and siB56 cells. BubR1 IPs from siB56 cells had similar and Pines, 2010). Thus, if aberrant SAC activity at the kinetochore levels of the BubR1 constitutive binding partner Bub3, but revealed leads to production of more MCC in siB56 cells, then cyclin A2 reductions in Cdc20, APC4, APC7 and APC8 association (Fig. 4A proteolysis will be delayed. To test this possibility, RPE1 compare lanes 4 and 5, quantified in Fig. 4B). The reduction in CCNA2Venus/+ siCtrl or siB56 cells were imaged before and after APC/C co-precipitation with BubR1 is consistent with reduced entry into mitosis. Nocodazole was included to eliminate indirect Cdc20 binding, as BubR1 association with APC/CCdc20 occurs effects on SAC activity due to defects in kinetochore–microtubule mainly through Cdc20 (Alfieri et al., 2016). Next, we asked whether attachment caused by B56 depletion (Foley et al., 2011). We found eliminating the binding of BubR1 to PP2AB56 is sufficient to reduce that cyclin-A2–Venus was degraded with similar kinetics in siCtrl APC/C–Cdc20 association. As outlined in Fig. 4C, we used siRNA and siB56 cells (Fig. 2F), suggesting that at the single cell level, to deplete endogenous BubR1 by 70% (Fig. 4D) and then there is no increase in the amount of MCC. In summary, we find no engineered cells to express localization and affinity purification evidence that the delay in mitotic exit in siB56 cells results from (LAP)-tagged BubR1 wild-type (WT) or a mutant in which the increased production or stabilization of the MCC. KARD had been deleted (ΔKARD) (Suijkerbuijk et al., 2012). We confirmed that BubR1-depleted cells were deficient in SAC PP2AB56 promotes APC/C–Cdc20 association activation and that expression of LAP–BubR1 restored SAC Interestingly, APC4 IPs from siB56 cells had reduced levels of activity (Fig. S2). LAP–BubR1ΔKARD did not interact with the Cdc20 and BubR1 (Fig. 3A, compare lanes 6 and 8; quantified in catalytic subunit of PP2A as expected (Kruse et al., 2013; Fig. 3B). As a control for APC/C assembly, we verified that Suijkerbuijk et al., 2012; Xu et al., 2013). However, LAP- association between APC4 and another APC/C subunit, APC7 (also BubR1ΔKARD bound to similar levels of Cdc20 and APC7 as known as ANAPC7), was unchanged. This decrease in Cdc20 and LAP–BubR1WT (Fig. 4E, compare lanes 4 and 5, quantified in B56 BubR1 binding to APC4 was rescued by RNAi-resistant B56ε, Fig. 4F). Thus, abrogating the association of BubR1 with PP2A Journal of Cell Science

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Fig. 2. B56 depletion does not increase the amount or stability of the mitotic checkpoint complex. (A) Schematic of experiment to isolate different pools of MCC from whole-cell lysates, including total MCC (Cdc20 IP), MCC bound to APC/C (co-precipitation in APC4 IP), and MCC free of APC4 [isolated by subjecting APC4-depleted supernatant (4S) to Cdc20 IP (Cdc204S IP)]. (B) Comparison of APC4 levels in siCtrl and siB56 whole-cell lysates versus APC4-depleted supernatant by quantitative western blotting. Numbers indicate the relative amount of APC4 in lysate versus APC4-depleted supernatant. *, non-specific band. (C) Whole-cell lysate (lanes 1 and 2) and IPs performed as outlined in A (lanes 3-8) were probed by western blotting. In B,C, solid vertical lines indicate that intervening lanes have been removed. (D,E) MCC is not stabilized in siB56 cells. Mitotic HeLa siCtrl and siB56 cells were incubated with MG132 and reversine or DMSO before lysis and then analyzed directly (D, lanes 1,2) or split for immunoprecipitation of Mad2 (D, lanes 3-10) and Cdc20 (E) and probed by western blotting. (F) Cyclin A proteolysis is not delayed in siB56 cells. RPE1 CCNA2Venus/+ siCtrl or siB56 cells in nocodazole were imaged live. (Left) Micrographs of Venus (cyclin A2) are shown with time (min) relative to nuclear envelope breakdown (NEBD). (Right) The fraction of cyclin-A2–Venus fluorescence remaining as a function of time in mitosis is plotted. Each line indicates a single cell. Scale bar: 5 μm. Results in B–F are representative of three independent experiments.

Ab, antibody; sup, supernatant; Trev, time before or after reversine addition. is insufficient to reduce the interaction of BubR1 with APC/CCdc20. Under identical inhibitor conditions, BubR1-depleted cells that had Moreover, Cdc20 IPs from BubR1-depleted cells rescued with WT been rescued with LAP–BubR1ΔKARD rapidly exited mitosis, with a and ΔKARD LAP–BubR1 constructs contained similar levels of median exit time of 16 min, which was indistinguishable from that APC4, APC7 and APC8 (Fig. 4G, compare lanes 4 and 5, quantified of BubR1-depleted cells that had been rescued with LAP–BubR1WT in Fig. 4H). Thus, we conclude that, unlike B56 depletion, (Fig. 4J). Based on these data, we conclude that the association of eliminating the association of PP2AB56 with BubR1 is insufficient PP2AB56 with BubR1 is not critical for promoting APC/CCdc20 to impair APC/CCdc20 assembly. However, we cannot exclude the assembly or exit from mitosis. Moreover, because the BubR1 possibility that the residual BubR1 after RNAi (Fig. 4D), which is KARD is required for PP2AB56 targeting to the kinetochore (Kruse too low to sustain mitotic arrest (Fig. S2), is nevertheless sufficient et al., 2013; Suijkerbuijk et al., 2012; Xu et al., 2013), our results for APC/CCdc20 assembly. imply that the cytoplasmic pool of PP2AB56 is sufficient for Our observation that the role of PP2AB56 in promoting APC/C– promoting mitotic exit. Cdc20 association does not require its interaction with BubR1 indicates that B56 depletion will yield a more severe effect on PP2AB56 interacts with APC/C independently of BubR1 mitotic exit than deletion of the BubR1 KARD. To test this In IPs of endogenous B56α, we observed co-precipitation of APC/C possibility, we scored the duration of mitotic arrest in cells that subunits, including APC1, APC4 and APC7, as well as of Cdc20 entered mitosis in the presence of nocodazole and reversine. As and BubR1 (Fig. 5A, compare lanes 3 and 4). Interestingly, while expected, inhibition of Mps1 induced rapid exit from mitosis in the association of B56α with Cdc20 and BubR1 was mitosis siCtrl cells with a median exit time of 15 min. By contrast, 75% of specific, as expected (Kruse et al., 2013), the interaction with APC/ siB56 cells remained arrested in mitosis after 100 min (Fig. 4I). C subunits was observed in IPs from mitotic and asynchronous Journal of Cell Science

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Fig. 3. PP2AB56 stimulates Cdc20 association with APC/C. (A–D) B56 depletion reduces the association of Cdc20 with APC/C. (A,B) HeLa Flp-In cells expressing doxycycline-inducible RNAi-resistant HA–FLAG–B56ε (B56εRNAi resis) were treated with siRNA, induced with doxycycline and collected by mitotic shake-off. (A) Lysates (lanes 1-4) and control IgG (lane 5) or APC4 IPs (lane 6-9) were analyzed by western blotting. (B) The experiment in A was performed three times and the results quantified. The value in siCtrl samples without B56ε induction was set to 1. (C,D) siCtrl and siB56 HeLa cells were collected by mitotic shake- off. (C) Lysates (lanes 1,2) and control IgG (lane 3) or APC3 (lane 4-5) IPs were probed by western blotting for the indicated proteins. (D) Quantification of three replicate experiments as described in C. (E) RPE1 cells were collected by mitotic shake-off, treated with DMSO or okadaic acid for 30 min and then used for IP of APC4 (lanes 3,4). (F,G) siB56 cells have less APC/C associated with Cdc20. (F) siCtrl and siB56 lysates (lanes 1,2) were subjected to Cdc20 (lanes 4,5) or control IgG (lane 3) IP. (G) The experiment in F was performed three times and quantified. Bars indicate mean±s.d. n.s., not significant (P>0.05); *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005, Student’s two-tailed t-test. cells (Fig. 5A, compare lanes 4 and 5, quantified in Fig. 5B). deletion of the KARD had no significant effect on the association Considering that BubR1 association with B56α was reduced by of B56α with Cdc20, APC4 and APC7, while APC1 association 93±7% (mean±s.d.) in asynchronous cells compared to mitotic cells was slightly increased (Fig. 5C, compare lanes 4 and 5, quantified in while association with APC1, APC4 and APC7 was unchanged, we Fig. 5D). From these results, we conclude that the APC/C and reasoned that B56α association with the APC/C might occur PP2AB56 interact constitutively and that this association does not independently of its association with BubR1. To test this possibility, depend on PP2AB56 targeting to BubR1. we performed B56α IPs in BubR1-depleted cells that had been Next, we asked if an LxxIxE docking site mediates PP2AB56 and WT ΔKARD rescued with LAP–BubR1 or LAP–BubR1 . Indeed, APC/C association. To this end, we compared the ability of B56α Journal of Cell Science

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Fig. 4. PP2AB56-dependent stimulation of APC/CCdc20 assembly does not require direct binding between B56 and BubR1. (A) BubR1 association with APC/C is reduced in siB56 cells. Mitotic siB56 or siCtrl HeLa cells were used for BubR1 (lanes 4,5) or control (lane 3) IgG IPs and analyzed by western blotting. (B) Quantification of three replicate experiments as in (A). (C–H) Deletion of the KARD in BubR1 does not alter APC/C–Cdc20 association. (C) Schematic for BubR1 depletion and rescue in HeLa cells. (D) BubR1-rescue lysates were analyzed alongside lysates from untreated cells by quantitative western blotting. Endogenous BubR1 signals were normalized to the actin signal, and the value in the untreated sample was set to 1. Solid line indicates intervening laneshave been cropped. (E–H) Lysates (lanes 1,2) and IPs (lanes 3-5) of GFP (E) or Cdc20 (G) were analyzed by western blotting. The experiments in E,G were performed three times and the quantifications are shown in F and H, respectively. (I,J) Comparison of mitotic exit delay in siB56 cells versus BubR1-rescue cells. siCtrl and siB56 cells (I) or BubR1-rescue cells (J) were incubated in nocodazole and reversine and imaged live. Plotted is the fraction of cells that exited mitosis as a function of time after mitotic entry. n, total number of cells analyzed from three independent experiments. Bars are mean±s.d. n.s., not significant (P>0.05); *P<0.05, **P<0.005, ***P<0.0005, Student’s two-tailed t-test.

WT and a point mutant (R222E) that abrogates binding to the docking is essential for the association between APC/C and LxxIxE motif (Hertz et al., 2016) to bind to APC/C. YFP-tagged PP2AB56. An LxxIxE motif has not been identified in human B56α WT or R222E expression was induced in HeLa cells, and APC/C subunits, and thus further work will be required to identify mitotic lysates were used for IP. We confirmed that the R222E the docking site for PP2AB56 on APC/C. mutation abolished BubR1 association but preserved PP2A catalytic subunit binding (Fig. 5E, compare lanes 4 and 5). Importantly, the Phosphorylation at Ser92 on Cdc20 controls its association association with Cdc20, APC4 and APC7 was reduced by the with the APC/C R222E mutation (Fig. 5E, compare lanes 4 and 5, quantified in We considered the possibility that increased phosphorylation of Cdc20 Fig. 5F), indicating that the region of B56α required for LxxIxE Cdc20 in siB56 cells might contribute to the defect in APC/C Journal of Cell Science

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Fig. 5. B56α interacts with the APC/C constitutively and independently of BubR1. (A,B) B56α co-precipitates with APC/C. (A) Mitotic (mit.) or asynchronous (asy.) HeLa cells were subjected to IP for B56α (lanes 4,5) or with control IgG (lane 3) and probed by western blotting for the indicated proteins. (B) Quantification of three replicate experiments as described in A. Values were normalized to those for the PP2A catalytic subunit (PP2A cat.). (C,D) Mitotic interaction of B56α and APC/C does not require the KARD of BubR1. BubR1 depletion and rescue was performed as described in Fig. 4C, and cells were subjected to IP for B56α (lanes 4,5) or with control IgG (lane 3) and probed by western blotting. (D) Quantification of three replicate experiments as described in C. (E,F) An LxxIxE motif mediates association of APC/C with B56α. Expression of YFP–B56α WT or an R222E mutant was induced by doxycycline in HeLa FLP-In lines, and mitotic cells were collected. (E) IPs for GFP (for YFP-tagged constructs; lanes 4,5) and control (lane 3) IPs were analyzed by western blotting. Asterisk indicates non- specific band. (F) Quantification of three replicate experiments described in E. Data are mean±s.d. n.s., not significant (P>0.05); *P<0.05, **P< 0.005, ***P<0.0005, ****P<0.00005, Student’s two-tailed t-test.

assembly. Indeed, phosphorylation of Cdc20 at sites adjacent to the therefore wanted to know whether Ser92 phosphorylation C-box, a motif essential for APC/C activation (Chang et al., 2014; modulates Cdc20 binding to the APC/C. A non-phosphorylatable Kimata et al., 2008), reduces Cdc20 binding to the APC/C (Hein Cdc20S92A mutant exhibited increased binding to APC/C subunits and Nilsson, 2016; Labit et al., 2012). To test this possibility, we compared to Cdc20WT or Cdc20S92E, a mutant that mimics utilized antibodies specific to Cdc20 that had been phosphorylated phosphorylation (Fig. 6D, compare lanes 5-7). Finally, we on Thr59 or Thr70 (Hein and Nilsson, 2016). We observed no combined B56 depletion with Cdc20 overexpression to determine increase in phosphorylation at either site in Cdc20 IPs from siB56 whether Cdc20S92A rescues the delay in mitotic exit in siB56 cells cells compared to those from siCtrl cells, although in the same (Fig. 6E). We determined that FLAG–Cdc20WT was expressed at experiment, siB56 cells exhibited decreased BubR1, APC4 and higher levels than FLAG–Cdc20S92A (Fig. 6F), precluding direct APC7 association (Fig. 6A, compare lanes 4 and 5, quantified in comparisons between the two cell lines. However, in both cell lines, Fig. 6C). Thus, reduced APC/C–Cdc20 association in siB56 cells B56 depletion delayed mitotic exit (Fig. 6G), indicating that the is not due to hyperphosphorylation of Thr59 and Thr70, although presence of Cdc20S92A is insufficient to rescue the mitotic exit we cannot exclude potential contributions from additional delays in siB56 cells. The lack of a rescue could arise from phosphorylation sites near the C-box of Cdc20. insufficient expression of FLAG–Cdc20, a requirement for the We confirmed that phosphorylation of Ser92 in Cdc20, a hydroxyl group on Ser92 of Cdc20, and/or contributions from other substrate of the kinase Plk1, is dephosphorylated by PP2AB56 PP2AB56 substrates towards mitotic exit. (Craney et al., 2016; Jia et al., 2016) (Fig. 6B, compare lanes 4 and 5, quantified in Fig. 6C). Mutations that abolish Ser92 DISCUSSION phosphorylation prevent delivery of Ube2S to APC/C and impair PP2AB56 is essential for mitosis, but because its specificity arises

APC/C activation in vitro (Craney et al., 2016; Jia et al., 2016). We through protein–protein interactions rather than through the Journal of Cell Science

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Fig. 6. Residue Ser92 on Cdc20 is a PP2AB56 substrate that influences Cdc20 binding to APC/C. (A,B) siCtrl and siB56 cells were collected by mitotic shake- off. Lysates (lanes 1,2), and control IgG (lane 3) or Cdc20 IPs (lanes 4,5) were analyzed by western blotting for the indicated proteins. (C) Quantification of three replicate experiments described in A,B. Data are mean±s.d. n.s., not significant (P>0.05), **P<0.005, Student’s two-tailed t-test. (D) Ser92 is a determinant of Cdc20 binding to APC/C. HeLa cells expressing doxycycline-inducible FLAG–Cdc20 wild type (WT), or the mutants S92A or S92E were collected by mitotic shake-off, lysed and analyzed directly (lanes 1-3) or used in a FLAG (lanes 5-7) or control IgG (lane 4) IP and probed for the indicated proteins by western blotting. (E–G) Cdc20S92A expression does not rescue mitotic exit delays in siB56 cells. (E) Schematic of B56 depletion and FLAG-Cdc20 induction in HeLa cells. (F) Mitotic siCtrl and siB56 cells expressing FLAG–Cdc20 constructs were harvested as outlined in E and analyzed alongside control mitotic HeLa lysates by western blotting. endog, endogenous. (G) Cells were imaged live and duration of mitosis was scored. The cumulative fraction of cells that exited mitosis as a function of time after mitotic entry is plotted. 50 cells were scored per condition. Experiments in D,F,G were performed in triplicate. (H) Schematic of PP2AB56- dependent stimulation of APC/C association with Cdc20. recognition of a consensus phosphorylation site, predicting its independent of BubR1 binding to PP2AB56. We identify residue substrates and functions during mitosis remains challenging. Here, Ser92 on Cdc20, a mitosis-specific Plk1 and PP2AB56 substrate we have analyzed the defects associated with mitotic exit delays in (Craney et al., 2016; Jia et al., 2016), as a determinant of APC/ siB56 cells. Previous work has established that siB56 cells are CCdc20 assembly. delayed in mitotic exit when Mps1 is inactivated in nocodazole- Substrate selectivity of the PP2AB56 holoenzyme arises, at least in treated cells (Espert et al., 2014; Nijenhuis et al., 2014). While we part, from the affinity of B56 subunits for the LxxIxE motif (Hertz confirm that B56 depletion causes increased kinetochore et al., 2016). We show that PP2AB56 associates constitutively with recruitment of SAC proteins in Mps1-inhibited cells, our data APC/C subunits and that this association depends on the ability of suggest that such changes may be insufficient to alter the level B56 subunits to recognize an LxxIxE motif. In mitosis, docking of and/or stability of the MCC. Rather, we find that PP2AB56 is PP2AB56 to the LxxIxE motif in the KARD of BubR1 localizes required to stabilize the association of Cdc20 with the APC/C, and PP2AB56 to the kinetochore, stabilizes kinetochore–microtubule in this way, promotes exit from mitosis (Fig. 6H). APC/C subunits attachments (Kruse et al., 2013; Suijkerbuijk et al., 2012; Xu et al., B56 associate with PP2A constitutively, and this association is 2013) and stimulates release of BubR1 from the kinetochore after Journal of Cell Science

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Mps1 inactivation (Espert et al., 2014; Nijenhuis et al., 2014). By concentrations: nocodazole (3.3 μM, except in Fig. 4I,J, used at 0.8 μM, contrast, we find that deletion of the BubR1 KARD has no effect on Sigma), MG132 (10 μM, Selleck Biochemicals, Houston, TX), reversine the association of Cdc20 with APC/C or on the timing of mitotic (1 μM, except Fig. 4I,J, used at 0.5 μM, Cayman Chemical, Ann Arbor, MI), B56 – RO-3306 (10 μM, Sigma), okadaic acid (1 μM, Santa Cruz Biotechnology, exit, suggesting that the role of PP2A in promoting APC/C μ Cdc20 association and exit from mitosis is likely to operate in Dallas, TX) and doxycycline (1 g/ml, Thermo Fisher Scientific). For siRNA transfection, Lipofectamine RNAi Max (Thermo Fisher parallel to its kinetochore-centered functions. Moreover, our Scientific) was used following the manufacturer’s reverse transfection findings imply that impaired SAC silencing at the kinetochore protocol. A non-targeting siRNA (GE Life Sciences, Marlborough, MA, cat. cannot account for the delay in mitotic exit in siB56 cells. Rather, # D-001810-01, sequence 5′-UGGUUUACAUGUCGACUAAUU-3′)was we propose that the critical dephosphorylation event(s) mediated by used for control transfections. The siB56 mix was prepared as described for PP2AB56 can proceed in the cytoplasm and converge on APC/ ‘pool two’ previously (Foley et al., 2011) omitting B56β siRNA, except as CCdc20 assembly and function. indicated in Fig. 1A-D. For microscopy analyses, 1.8×105 RPE1 cells were Our results do not preclude a role for the SAC or kinetochore transfected with 100 pmol siRNA and 5.0 µl Lipofectamine RNAi Max. For targeting of other factors in the production of the preceding HeLa cells, the transfection reagents were halved. For biochemical 6 phosphorylation events that limit APC/CCdc20 assembly. Indeed, experiments, 5×10 RPE1 cells were transfected with 1600 pmol siRNA Plk1, which phosphorylates Ser92 on Cdc20 (Craney et al., 2016; and 80 µl Lipofectamine RNAi Max. For siRNA treatment for biochemical analyses in HeLa cells, 5×106 cells were transfected with 800 pmol siRNA Jia et al., 2016), cooperates with Mps1 to activate the SAC at and 40 µl Lipofectamine RNAi Max. Cells were imaged, fixed or harvested unattached kinetochores (Espeut et al., 2015; von Schubert et al., at 42-48 h post transfection. Mitotic cells were collected by manual 2015). Moreover, both Cdc20 and the APC/C localize to unattached detachment following 14-16 h of incubation in nocodazole. The mitotic kinetochores (Acquaviva et al., 2004; Kallio et al., 2002). index was enumerated by scoring individual cells for the presence or Therefore, it is possible that the relevant phosphorylation events absence of mitotic chromatin (condensed chromosomes) and was typically that reduce Cdc20 binding to the APC/C, which likely include Plk1- >90% in siCtrl and siB56 samples. BubR1 depletion and rescue experiments dependent phosphorylation of Cdc20 at Ser92, could be generated at were performed as described previously (Suijkerbuijk et al., 2012). RNAi- Venus/+ unattached kinetochores through SAC signaling. Moreover, we have resistant-B56α-expressing RPE1 cells (Foley et al., 2011), CCNA2 and CCNB1Venus/+ RPE1 cells (Collin et al., 2013), and HeLa cells previously shown that Plk1 targeting to the kinetochore is increased – in siB56 cells (Foley et al., 2011). Thus, it is possible that the expressing FLAG Cdc20 wild type or the S92A and S92E mutants (Jia et al., 2016) have been described previously. increase in phosphorylation at Ser92 that we observe in siB56 cells arises from both increased Plk1 levels at the kinetochore and decreased PP2AB56 levels in the cytoplasm. Plasmids ε B56 subunits target PP2A phosphatase activity to substrates to An RNAi-resistant B56 expression construct was generated using site- stimulate exit from mitosis in multiple ways, including through directed mutagenesis of a PPP2R5E Gateway entry clone (Foley et al., 2011). APC/CCdc20 assembly, as we show here, as well as by E2 ubiquitin- conjugating enzyme recruitment to the APC/C (Craney et al., 2016) and through SAC signaling at the kinetochore (Espert et al., 2014; Antibodies Nijenhuis et al., 2014). A key challenge will be to discern how these All antibodies were used at 1 µg/ml for immunofluorescence, 0.1 µg/ml for roles intersect synergistically to exert temporal control over western blotting, and 2-4 µg of IgG was used per immunoprecipitation unless otherwise specified. Antibodies were generated using peptide chromosome segregation alongside other mitotic functions of α B56 – sequences from APC4, BubR1 and B56 . Peptides were synthesized by PP2A , which include stabilization of kinetochore microtubule EZBiolab, Carmel, IN (APC4: CEIVIKVEKLDPELDS; BubR1: attachment (Foley et al., 2011) and preservation of sister chromatid CVKKEGGALSEAMSLE) or by Tufts University, Medford, MA (B56α: cohesion (Chen et al., 2007; Kitajima et al., 2006; Riedel et al., CKKALEKQNSAYNMHSILSNTSAE) to 90% purity. 4 mg of peptide 2006; Tang et al., 2006). Importantly, the assembly of PP2AB56 was coupled to maleimide-activated keyhole limpet hemocyanin (Thermo holoenzymes is predicted to be disrupted by recurrent cancer- Fisher Scientific). Custom antibodies were raised against recombinant associated heterozygous missense mutations in the scaffold subunit GST–GFP. Antigens were injected into New Zealand White rabbits using an of PP2A (Cho and Xu, 2007; Xu et al., 2006). These mutations are institutionally approved protocol and animal-care facility (Pocono Rabbit associated with tumors that undergo whole genome duplication Farm and Laboratory, Canadensis, PA). For peptide-reactive antibodies, sera (Zack et al., 2013), and it will be important to identify if any of the in 1× PBS were loaded onto sulfo-link columns (Thermo Fisher Scientific) mitotic functions of PP2AB56 are sensitive to these changes. conjugated to the immunizing peptide. For antibodies against GFP, serum was loaded onto a HiTrap NHS-activated high-pressure column (GE Life Sciences) coupled to GST–GFP. After washing with PBS, antibodies were MATERIALS AND METHODS eluted with 0.2 M glycine, pH 2.5 and subsequently neutralized with Tris- Cell culture, transfection and inhibitor treatments HCl pH 8.0, followed by dialysis into PBS. A custom antibody against B56ε Cells were grown at 37°C in a humidified atmosphere with 5% CO2 in (peptide sequence CRGLRRDGIIPT) was generated by Bethyl Dulbecco’s modified Eagle’s medium (DMEM) (HeLa, AmphoPack-293) Laboratories, Montgomery, TX. To validate custom antibodies for use in or 1:1 DMEM:F12 (RPE1) supplemented with 10% fetal bovine serum, IP, we confirmed that pre-incubation of the antibody with the immunizing penicillin-streptomycin, non-essential amino acids and L-glutamine. RPE1 peptide blocked IP of the target protein. To validate the anti-GFP antibody and HeLa cell lines were authenticated annually by short tandem repeat for western blotting, we confirmed that the antibody recognized analysis and tested for mycoplasma contamination every 6 months. For recombinant GFP protein and further confirmed its ability to detect immunofluorescence microscopy, cells were grown on no. 1.5 glass induction of GFP- or YFP-tagged proteins in human cells. For antibodies coverslips (Thermo Fisher Scientific) coated with poly-D-lysine (Sigma). against B56α and B56ε, which were also used for western blotting, we For live imaging, cells were grown in glass-bottom dishes with no. 1.5 cover confirmed that the signal attributed to the target protein was reduced by glass (Cellvis, Mountain View, CA). RPE1 cells lines stably expressing gene-specific RNAi. H2B–mCherry were generated as described previously (Foley et al., 2011). Commercial antibodies used in this study include those against: α-tubulin RNAi-resistant LAP–BubR1 WT and ΔKARD, and RNAi-resistant B56ε (mouse DM1α–FITC conjugate; Sigma, F2168); β-actin (Sigma, A5316); were introduced into HeLa FLP-In cells by site-specific recombination. The APC1 (Bethyl Laboratories, A301-653A); APC3 (BD Biosciences, 610455, following chemicals were dissolved in DMSO and used at the indicated western blot only); APC7 (Bethyl Laboratories, A302-551A); APC8 Journal of Cell Science

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(Bethyl Laboratories, A301-182A); BubR1 (BD Transduction Laboratories, England BioLabs, Ipswich, MA) for 30 min at 30°C. Proteins were resolved 612503; immunofluorescence and western blot only); PP2A catalytic by SDS-PAGE and transferred to nitrocellulose for western blotting via subunit (BD Transduction Laboratories, 610555); PP2A scaffold (Santa chemiluminescence using an ImageQuant LAS500 (GE Life Sciences). Cruz Biotechnology, sc-6112); Mad2 (Bethyl Laboratories, A300-301A); Intensity measurements from western blots were performed in Fiji. For B56γ (Bethyl Laboratories, A303-814A); B56δ (Bethyl Laboratories, presentation of input data, the input lanes were cropped and scaled to the A301-098A); and human CREST antiserum (Immunovision, Springsdale, minimum and maximum pixel intensity in the input samples using Fiji. The AR, HCT-0100; 1:5000). Antibodies against APC3 (used for IP only). IP lanes were processed in the same manner. Thus, input and IP samples are APC4 (used for western blot only) and Cdc20 phosphorylated at Thr59 and scaled differently. To indicate this, the input and IP portions of the blot are Thr70 were gifts from Jakob Nilsson (University of Copenhagen, Denmark) separated in the figure panels. Cropped and scaled images were imported (Hein and Nilsson, 2016). The antibody against Cdc20 phosphorylated at into Illustrator (Adobe, San Jose, CA) for figure assembly. Ser92 was a gift from Hongtao Yu (University of Texas Southwestern Medical Center, Dallas, TX) (Jia et al., 2016). Acknowledgements We thank Prasad Jallepalli for scientific discussion and critical reading of the Immunofluorescence microscopy manuscript and Geert Kops, Jakob Nilsson, Jonathon Pines and Hongtao Yu for reagents. Cells on coverslips were fixed and permeabilized by a 5 min incubation at 37°C in 20 mM PIPES, pH 6.8, 10 mM EGTA, 1 mM MgCl ,4% 2 Competing interests paraformaldehyde and 0.2% Triton X-100. Coverslips were then washed The authors declare no competing or financial interests. with TBS+0.1% Triton X-100 and blocked in 2% donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA) for 30 min and stained. Author contributions After washing in TBS+0.1% Triton X-100, samples were stained with Conceptualization: E.A.F.; Formal analysis: E.A.F., V.R.-B., H.K., S.D.; fluorescence-conjugated secondary antibodies (Jackson Immunoresearch Investigation: E.A.F., S.J.L., V.R.-B., H.K., S.D.; Writing - original draft: E.A.F.; Laboratories). Samples were mounted onto slides in Prolong Gold with Writing - review & editing: E.A.F.; Visualization: E.A.F.; Supervision: E.A.F.; Funding DAPI (Thermo Fisher Scientific) and sealed with nail polish. Samples were acquisition: E.A.F. imaged on DeltaVision Image Restoration System (GE Healthcare, Chicago, IL) based on an Olympus IX-70 microscope with a 100×1.4 NA Funding oil objective and a CoolSnap QE cooled CCD camera (Photometrics, This work was supported by a Gerstner Young Investigator Award to E.A.F. the Tucson, Arizona). z-stacks were acquired with 0.2 μm spacing. Image Geoffrey Beene Cancer Research Center of Memorial Sloan Kettering Cancer projections, crops and measurements were performed in Fiji (Schindelin Center, and the Functional Genomics Initiative at Memorial Sloan Kettering Cancer Center. We acknowledge the Memorial Sloan Kettering Cancer Center Support et al., 2012). Kinetochore regions were defined by a CREST antibody signal, Grant (P30 CA008748) from the National Cancer Institute. Deposited in PMC for and corresponding regions in other channels were used to calculate the release after 12 months. average kinetochore intensity signal per plane. Local background was defined by the DNA region minus the kinetochore region and was subtracted Supplementary information from the kinetochore signal. For each cell, the kinetochore signal was Supplementary information available online at averaged over all z-slices. http://jcs.biologists.org/lookup/doi/10.1242/jcs.201608.supplemental

Live-cell imaging References Cells in glass-bottom dishes were mounted on a Nikon Eclipse TiE/B Acquaviva, C., Herzog, F., Kraft, C. and Pines, J. (2004). The anaphase microscope operated through NIS Elements software (Nikon, Melville, promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint. Nat. Cell Biol. 6, 892-898. NY), equipped with 20×0.75 NA and 40×1.3 NA air objectives, a piezo Alfieri, C., Chang, L., Zhang, Z., Yang, J., Maslen, S., Skehel, M. and Barford, D. stage, Perfect Focus, a spinning disk confocal head (Yokogawa, Sugar Land, (2016). Molecular basis of APC/C regulation by the spindle assembly checkpoint. TX) with 488 nm laser line and EMCCD and sCMOS cameras. Cells were Nature 536, 431-436. maintained in an environmental enclosure with temperature-controlled stage Bastos, R. N., Cundell, M. J. and Barr, F. A. (2014). KIF4A and PP2A-B56 form a spatially restricted feedback loop opposing Aurora B at the anaphase central with 5% CO2 delivery (Tokai Hit, Fujinomiya, Japan). For mitotic timing measurements, cells were imaged at 20× by differential interference contrast spindle. J. Cell Biol. 207, 683-693. (DIC) imaging, and/or by fluorescence imaging. A minimum of 30 cells per Chang, L., Zhang, Z., Yang, J., McLaughlin, S. H. and Barford, D. (2014). – Molecular architecture and mechanism of the anaphase-promoting complex. condition was analyzed in each experiment. For cyclin-A2 Venus imaging, Nature 513, 388-393. cells in nocodazole were imaged at 20× magnification by confocal Chao, W. C. H., Kulkarni, K., Zhang, Z., Kong, E. H. and Barford, D. (2012). microscopy. Two z-planes were taken with 5 μm spacing every 4 min. For Structure of the mitotic checkpoint complex. Nature 484, 208-213. cyclin-B1–Venus imaging, cells in nocodazole were visualized at 40× Chen, F., Archambault, V., Kar, A., Lio, P., D’Avino, P. P., Sinka, R., Lilley, K., magnification by confocal microscopy and DIC before and after reversine Laue, E. D., Deak, P., Capalbo, L. et al. (2007). Multiple protein phosphatases addition. Eight to ten cells per condition were analyzed per experiment. The are required for mitosis in Drosophila. Curr. Biol. 17, 293-303. Cho, U. S. and Xu, W. (2007). Crystal structure of a protein phosphatase 2A non-cellular background fluorescence signal was subtracted and the mean μ heterotrimeric holoenzyme. Nature 445, 53-57. fluorescence intensity of a 4×4 m region was measured. Values were Chung, E. and Chen, R.-H. (2003). Phosphorylation of Cdc20 is required for its normalized to the fluorescence intensity of the first image after nuclear inhibition by the spindle checkpoint. Nat. Cell Biol. 5, 748-753. envelope breakdown or the image taken before reversine addition. Collin, P., Nashchekina, O., Walker, R. and Pines, J. (2013). The spindle assembly checkpoint works like a rheostat rather than a toggle switch. Nat. Cell Cell lysis and immunoprecipitation Biol. 15, 1378-1385. Lysates were prepared by suspension in buffer B (30 mM HEPES, pH 7.8, Craney, A., Kelly, A., Jia, L., Fedrigo, I., Yu, H. and Rape, M. (2016). Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation. Proc. 140 mM NaCl, 6 mM MgCl2, 5% glycerol) at 4°C supplemented with Natl. Acad. Sci. USA 113, 1540-1545. 2 mM DTT, 1% ProBlock Gold Mammalian Protease Inhibitor Cocktail D’Angiolella, V., Mari, C., Nocera, D., Rametti, L. and Grieco, D. (2003). The (GoldBio Technology, St. Louis, MO) and 1× PhosSTOP (Roche, spindle checkpoint requires cyclin-dependent kinase activity. Genes Dev. 17, Indianapolis, IN) followed by nitrogen cavitation (Parr Instruments, 2520-2525. Moline, IL) for 5 min at 2000 psi on ice, and centrifugation at 20,000 g Di Fiore, B. and Pines, J. (2010). How cyclin A destruction escapes the spindle for 15 min. Proteins were immunoprecipitated with antibody bound to assembly checkpoint. J. Cell Biol. 190, 501-509. Protein A or Protein G Dynabeads (Thermo Fisher Scientific) for 45-60 min Dick, A. E. and Gerlich, D. W. (2013). Kinetic framework of spindle assembly checkpoint signalling. Nat. Cell Biol. 15, 1370-1377. at 4°C, and washed three times with buffer B at 4°C. In Fig. 6F, cells were Espert, A., Uluocak, P., Bastos, R. N., Mangat, D., Graab, P. and Gruneberg, U. suspended in 50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, (2014). PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes

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1770 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1760-1771 doi:10.1242/jcs.201608

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