Recovery from spindle checkpoint-mediated arrest requires a novel Dnt1-dependent APC/C activation mechanism.

The activated spindle assembly checkpoint (SAC) potently inhibits the anaphase-promoting complex/cyclosome (APC/C) to ensure accurate chromosome segregation at anaphase. Early studies have recognized that the SAC should be silenced within minutes to enable rapid APC/C activation and synchronous segregation of chromosomes once all kinetochores are properly attached, but the underlying silencers are still being elucidated. Here, we report that the timely silencing of SAC in fission yeast requires dnt1+, which causes severe thiabendazole (TBZ) sensitivity and increased rate of lagging chromosomes when deleted. The absence of Dnt1 results in prolonged inhibitory binding of mitotic checkpoint complex (MCC) to APC/C and attenuated protein levels of Slp1Cdc20, consequently slows the degradation of cyclin B and securin, and eventually delays anaphase entry in cells released from SAC activation. Interestingly, Dnt1 physically associates with APC/C upon SAC activation. We propose that this association may fend off excessive and prolonged MCC binding to APC/C and help to maintain Slp1Cdc20 stability. This may allow a subset of APC/C to retain activity, which ensures rapid anaphase onset and mitotic exit once SAC is inactivated. Therefore, our study uncovered a new player in dictating the timing and efficacy of APC/C activation, which is actively required for maintaining cell viability upon recovery from the inhibition of APC/C by spindle checkpoint.

Introduction

The eukaryotic cell cycle comprises two major alternate events, chromosome duplication during interphase and subsequent chromosome segregation during mitosis, usually with extremely high fidelity. When errors do occur, they can have catastrophic consequences, including cell death or genome instabilities. In late mitosis, chromosome segregation and anaphase onset are initiated through the action of a 1.2 MDa multi-subunit E3 ubiquitin ligase known as the anaphase-promoting complex or cyclosome (APC/C) [1]. Most of the APC/C subunits are essential for viability and are conserved in all eukaryotes from yeast to humans [2,3]. To fulfill its proper functions during mitosis, APC/C needs to cooperate with at least two ubiquitin-conjugating (E2) enzymes and one essential co-activator, Cdc20 (Slp1 in the fission yeast Schizosaccharomyces pombe), to recruit and ubiquitylate substrates for proteasomal degradation [4-8]. Securin (Cut2 in fission yeast) and cyclin B (Cdc13 in fission yeast) are two major APC/C substrates, and their polyubiquitylation and degradation are critical for anaphase onset and chromosome segregation [9]. In humans, deregulation of these control mechanisms and altered activity of the APC/C can lead to severe mitotic defects and genome instabilities and has been associated with the development of various human cancer types [10-14].

The spindle assembly checkpoint (SAC) is an intricate surveillance mechanism that prolongs mitosis until all chromosomes achieve correct bipolar attachments to spindle microtubules. The core components of the SAC, including Mad1, Mad2, BubR1 (also known as Mad3 in yeasts and worms), Bub1, Bub3 and Mps1 (Mph1 in fission yeast), accumulate on unattached kinetochores and start a signaling cascade that ultimately inhibits Cdc20 [4,15,16]. The mitotic checkpoint complex (MCC) is composed of 3–4 core proteins (Cdc20-BubR1/Mad3-Mad2 with or without Bub3 depending on species) and has been found to be the most potent inhibitor of the APC/C prior to anaphase [4,17,18]. Recent biochemical and structural studies revealed that the human and fission yeast MCCs bind two Cdc20 molecules, one (Cdc20MCC) through cooperative binding to Mad2 and Mad3/BubR1 (forming the “core MCC”) and the other (Cdc20APC/C) through additional binding motifs in BubR1/Mad3 [19-23].

Although it has been well established that the production of MCC and SAC signaling must be inactivated once chromosomes are properly aligned on the spindle before anaphase onset, the molecular mechanisms of SAC inactivation remain obscure [4,17]. So far, about six proteins including p31comet, the AAA+ ATPase TRIP13, UbcH10, Spindly, CUEDC2 and phosphatase PP2A have been suggested to be involved in checkpoint inactivation in mammalian cells [24-31]. p31comet, TRIP13, CUEDC2 and Spindly ensure timely spindle checkpoint silencing subsequent to kinetochore attachment through promoting the release of Mad2 from MCC or stripping of Mad2 from the kinetochore, respectively [27,28,32-38], while PP2A inactivates SAC by reversing the Mps1-mediated Knl1 phosphorylation necessary for Bub1/BubR1 recruitment [29]. By contrast, the spindle checkpoint-silencing mechanisms in fungi remains less well explored, limiting the full understanding about this key cellular process. In both budding and fission yeast, type 1 phosphatase PP1 (Dis2 in fission yeast) is required for spindle checkpoint silencing by opposing Aurora B kinase, although their targeted substrates still remain unidentified [39-41]. Whether other proteins and mechanisms are involved in SAC silencing in yeast is currently unclear.

Fission yeast Dnt1 accumulates mainly in the nucleolus throughout the entire cell cycle and was originally identified in a genetic screen for suppressors of the cytokinesis checkpoint defects in the septation initiation network (SIN) mutants [42]. This suppression was later attributed to a mechanism that is mediated by the Cdk1 regulator Wee1 kinase [43]. Dnt1 also appears as a negative regulator of Dma1 in early mitosis [44]. Dma1, the checkpoint protein and E3 ubiquitin ligase, is a distinct spindle checkpoint protein and plays an important role in delaying cytokinesis by inhibiting the SIN when chromosomes are not attached to the mitotic spindle [45-47]. In this study, we identified Dnt1 as a novel factor involved in inhibiting excessive and prolonged MCC-APC/C association and promoting Slp1Cdc20 stability, and thus it ensures timely spindle checkpoint inactivation, which is essentially required for maintaining cell viability upon recovery from the inhibition of APC/C by checkpoint.

Results

Dnt1 is involved in facilitating proper mitotic chromosome segregation

In the course of our characterizing Dnt1 as a negative regulator of Dma1 in early mitosis [44], we noticed that dnt1Δ cells were extremely sensitive to microtubule-destabilizing drug thiabendazole (TBZ), and deletion of dma1 in these cells only slightly reversed this phenotype (Fig 1A). Since the disruption of genes participating in chromosome segregation, such as the kinetochore- or spindle checkpoint-related genes, usually results in TBZ sensitivity [48,49], we assumed that dnt1 gene might have dma1-irrelevant function that is essential for chromosome segregation.

Fig 1

Characterization on the involvement of Dnt1 in facilitating proper mitotic chromosome segregation.

(A) Tenfold serial dilution analyses of the indicated yeast strains grown on the indicated media to measure the TBZ sensitivity. (B) Minichromosome loss rate per division was measured in strains bearing the Ch16 (ade6-M216) minichromosome and ade6-M210 allele (n > 8,000). (C) (Left) Chromosome segregation was observed in anaphase in the indicated cells carrying mCherry-tagged α-tubulin (atb2) (red) after being fixed and stained with DAPI (blue). (Right) Quantitative analysis of chromosome missegregation and lagging chromosome phenotypes. n, numbers of anaphase cells analyzed. Scale bar, 5 μm. (D-F) Anaphase B is slightly delayed in dnt1Δ cells. (D) Time-lapse microscopy of a wild-type and a dnt1Δ cell carrying ndc80-GFP and sad1-mCherry during mitosis. Images were acquired at 2-min intervals. Arrows indicate a lagging chromosome. The enlarged image corresponding to the time frame indicated by red dot is shown in inset. Scale bar, 5 μm. (E) Distance between SPBs (SPB to SPB, marked by Sad1-mCherry) was measured at 2-min intervals. Each line represents data collected from an individual cell. Dashed lines indicate 3 μm, which roughly marks the timing for start of anaphase B in most cases. (F) Box-and-whiskers representation of anaphase B (during which pole-to-pole distance increases with separated sister chromatids) duration, in which boxes indicates median and upper and lower quartile and whiskers indicates range of data. The data were extracted and quantified from spindle dynamics measurements in (D). ****, p<0.0001.

Consistent with this assumption, we found that dnt1Δ cells lost minichromosomes (Ch16, ade6-M216) at an elevated rate that is almost 100 times higher than that of the wild-type (Fig 1B), and displayed increased frequency of lagging chromosomes and chromosome mis-segregation at mitotic anaphase (Fig 1C). By following the kinetochore separation and spindle dynamics during mitosis under time-lapse microscopy, we found that dnt1Δ cells spent almost the same length of time at late prometaphase/metaphase and anaphase A as wild-type cells except for the occasionally observed lagging chromosomes (2 cases in 14 dnt1Δ cells but 0 in 17 wild-type cells) at early anaphase, but dnt1Δ cells stayed for extended length of time at anaphase B (Fig 1D–1F). These data strongly suggested that Dnt1 is involved in facilitating proper chromosome segregation.

Chromosome segregation is a precisely regulated process involving many proteins, including kinetochore proteins, monopolins, cohesins, chromosome passenger proteins, centromeric heterochromatin proteins, microtubule-binding proteins, and regulators of kinetochore-microtubule attachment [50]. In order to dissect the mechanism behind how Dnt1 is involved in maintaining the fidelity of chromosome segregation, we systematically tested the genetic interactions between dnt1Δ and some representative mutants that have been previously reported to cause chromosome missegregation. Strikingly, dnt1Δ showed mild to strong negative genetic interactions with almost all these mutants (S1 Fig), indicating that Dnt1 became essential when any of these proteins was absent or mutated. Thus, Dnt1 should be involved in faithful mitotic chromosome segregation in a previously unrecognized manner.

Dnt1 is dispensable for activating the SAC in the absence of kinetochore-microtubule attachment or tension

The spindle checkpoint is another safeguard mechanism that ensures proper chromosome segregation, which is rapidly activated when the kinetochore-spindle microtubule attachment or the tension generated by this attachment is absent or compromised [16]. We then examined whether the SAC is properly activated in dnt1Δ cells when the kinetochore-spindle microtubule attachment or the tension was compromised by either the cold-sensitive β-tubulin mutation nda3-KM311 [51] or the temperature-sensitive cohesin subunit mutation psc3-1T [52,53], respectively. In these two experimental set-ups, the accumulation of two major APC/C substrates cyclin B (Cdc13-GFP) and securin (Cut2-GFP) at SPBs or within nuclei, respectively, served as an indicator of SAC activation and mitotic arrest (S2 Fig). Our quantified data showed that the Cdc13-GFP or Cut2-GFP were accumulated at SPBs or within nuclei in nda3-KM311 or pst3-1T cells, respectively, with indistinguishable rate in the presence and absence of dnt1 (S2B and S2D Fig), indicating that Dnt1 is largely dispensable for activating SAC in the absence of either attachment or tension.

Dnt1 facilitates efficient anaphase initiation upon SAC inactivation

The foregoing results rendered us to hypothesize that Dnt1 might have a role in turning off the spindle checkpoint in mitosis. To explore this, we adopted two well-established SAC silencing assays [39,54]. In these assays, the SAC was first robustly activated by the nda3-KM311 mutant simultaneously carrying ark1-as3 and then inactivated by addition of ATP analogue 1-NM-PP1 or simply by shifting mitotically arrested cells back to permissive temperature (30°C) as previously described (Figs 2A and S3A). Because Cdc13 (cyclin B) and Cut2 (securin) localize to the spindle pole bodies (SPBs) or nucleus, respectively, in early mitosis and should be degraded by APC/C to promote metaphase-anaphase transition, the disappearance rate of Cdc13-GFP spot or nuclear Cut2-GFP after 1-NM-PP1 addition or shifting back to 30°C reflects the SAC inactivation efficacy (Figs 2B and S4A). We found that dnt1Δ cells retained high amounts of SPB-localized Cdc13-GFP and nuclear Cut2-GFP for much prolonged period compared to wild-type cells, almost to the same degree as previously identified SAC-inactivation defective mutant dis2Δ [39] (Figs 2C, S3B, and S4B). Also, the total cellular protein of Cdc13 or Cut2 was indeed degraded much slower in dnt1Δ cells than in wild-type cells recovered from nda3-mediated checkpoint arrest (Figs 2D, 2E and S4B). All above data suggested that Dnt1 is required for the timely inactivation of SAC to efficiently initiate anaphase.

Fig 2

Dnt1 facilitates timely degradation of Cyclin B and dissociation of Mad2 and Mad3 from APC/C complex upon SAC inactivation.

(A) Schematic depiction of the experiment design for (B-G and I). (B-E) Dnt1 is required for timely degradation of Cyclin B upon SAC inactivation. Example pictures of cells with Cdc13-GFP signals enriched or disappeared at spindle pole bodies (SPBs) are shown in (B). The percentage of cells with Cdc13-GFP on SPBs was assessed at each time point after shift to 30°C (C), which served as an indicator for mitotic exit. Total protein levels of Cdc13-GFP were detected by Western blotting (D) and normalized to those of total Cdc2 at each time point, with the relative ratio between Cdc13-GFP and Cdc2 at 0 min set as 1.0 (E). Scale bar, 5 μm. (F-G) Dissociation of the Mad2 and Mad3 checkpoint proteins from the APC/C during mitotic arrest-and-release is delayed in dnt1Δ cells. The association of Mad2 and Mad3 to Apc4/Lid1 was assessed by immunoprecipitation of Apc4/Lid1-TAP and Western blot (F). The amount of co-immunoprecipitated Mad2 and Mad3 was normalized to those of total immunoprecipitated Apc4/Lid1 at each time point, with the relative ratio between Mad2-GFP or Mad3-GFP and Apc4/Lid1-TAP at 0 min set as 1.0 (G). (H) Schematic summary of results shown in C-G. Prolonged MCC-APC/C association in dnt1Δ cells anti-correlates with decelerated anaphase entry and mitotic exit upon spindle checkpoint inactivation. (I) Depletion of SAC signaling by deletions of mad2 or bub1 abrogates the delayed SAC inactivation after release from checkpoint arrest in dnt1Δ cells. Cells of indicated strains bearing Cdc13-GFP were treated and assessed as in (B and C). The experiments were repeated two (D-G) or three (C and I) times. Error bars correspond to standard deviation (SD).

Dnt1 is required for timely dissociation of MCC from APC/C during SAC inactivation

In fission yeast, the key SAC components Mad2 and Mad3 and one molecule of Slp1Cdc20 form mitotic checkpoint complex (MCC) which binds to APC/C through another molecule of Slp1Cdc20 upon checkpoint arrest, and the recovery from mitotic arrest accompanies the loss of MCC-APC/C binding [22,39,55,56]. To examine whether delayed anaphase initiation upon recovery from mitotic arrest in dnt1Δ cells was due to persistent MCC-APC/C binding, we analyzed the rate of dissociation of the MCC from the APC/C by immunoprecipitations of the APC/C subunit Apc4/Lid1 from mitosis-exiting cells. Indeed, the levels of MCC bound to the APC/C in dnt1Δ cells stayed high for longer period than in wild-type cells (Fig 2F and 2G), suggesting that Dnt1 functions for timely and efficient MCC-APC/C dissociation and spindle checkpoint inactivation (Fig 2H), which is required for activating APC/C to degrade Cdc13 and Cut2. Deletions of mad2 or bub1 significantly lowered the percentage of dnt1Δ cells with Cdc13 at SPBs upon nda3-mediated checkpoint activation (Fig 2I), indicating that sustained SAC activation and consequent prometaphase- or metaphase-arrest in the absence of Dnt1 relies on prior SAC activation.

Dnt1 positively regulates the protein levels of Slp1Cdc20

Given that MCC functions as a potent inhibitor of APC/C upon spindle checkpoint activation and Dnt1 is required to promote timely dissociation of MCC from APC/C, we were suspicious that one direct consequence of the prolonged MCC-APC/C association in dnt1Δ cells might be the inhibition of APC/C activity. Consistent with this assumption, we found that dnt1Δ enhanced the growth defects of some temperature-sensitive mutants of essential APC/C subunits, such as nuc2-663, cut9-234, cut20-100 and cut23-547, though dnt1Δ did not have any negative genetic interactions with two other APC/C mutants apc15Δ and slp1-mr63, both are defective in spindle checkpoint arrest [55, 57] (Fig 3A). Most strikingly, we observed that dnt1Δ rescued slp1-362 surprisingly well (Fig 3A). This specific genetic interaction suggested a possible role of Dnt1 in MCC-APC/C dissociation through regulating Slp1Cdc20. To test this hypothesis, we examined the protein levels of the full-length Slp1Cdc20 in nda3-KM311 dnt1 and nda3-KM311 dnt1Δ cells after being released from metaphase-arrest. Surprisingly, Slp1Cdc20 was slightly, but appreciably and reproducibly, less abundant (ranging from roughly 20% to 50% at different time points) in dnt1Δ cells than in wild-type cells (Fig 3B), suggesting Dnt1 may indeed positively regulate the levels of intact Slp1Cdc20. In addition, this regulation of Slp1Cdc20 stability by Dnt1 is Dma1-independent, as the dnt1Δ dma1Δ double mutant has a similar level and degradation profile of Slp1Cdc20 as dnt1Δ single mutant (S5 Fig).

Fig 3

Dnt1 is required for maintaining the protein levels of Slp1Cdc20 upon SAC activation.

(A) dnt1Δ rescues the temperature-sensitivity of slp1-362 but not other loss-of-function APC/C mutants. Serial dilutions (10-fold) of the indicated strains were spotted on YE plates and incubated at the indicated temperatures. Note that temperature-sensitivity of most loss-of-function APC/C mutants is exacerbated by dnt1Δ, but dnt1Δ rescues slp1-362. In addition, dnt1Δ does not have any negative genetic interactions with two other APC/C mutants, apc15Δ and slp1-mr63, both are defective in spindle checkpoint arrest. (B) Slp1Cdc20 levels are slightly reduced in dnt1Δ cells compared to wild-type cells during metaphase arrest and SAC inactivation. Strains with indicated genotypes were grown, treated and synchronized as in Fig 2A. Mid-log phase samples at 30°C were also collected as asynchronous cultures (Asyn.). (Left) Samples were subjected to Western blot analyses using anti-Slp1 and anti-Cdc2 antibodies. (Right) Slp1Cdc20 levels were normalized to those of total Cdc2 at each time point, with the relative ratio between Slp1Cdc20 and Cdc2 at time 0 min in nda3-KM311 set as 1.0. (C) Schematic depiction of the genomic positions of endogenous slp1 locus and ectopic slp1 at lys1 on chromosome I and at leu1 on chromosome II. Red bars, two centromeres 1 and 2. (D) Western blot analyses of Slp1Cdc20 protein levels in metaphase-arrested cells. Slp1Cdc20 levels were normalized to those of total Cdc2 for each sample, with the relative ratio between Slp1Cdc20 and Cdc2 at 0 min in nda3-KM311 without extra copies of slp1 set as 1.0. Note that in the strain carrying both endogenous slp1 and lys1Δ::slp1, Slp1Cdc20 level is close to 2.5 instead of 2 times of endogenous level, most likely due to its flanking adh1 terminator sequence. Error bars correspond to standard deviation. p values were calculated against the strain of nda3-KM311 without extra copies of slp1. ***, p<0.001; **, p<0.01; n.s., not significant. (E) Elevated Slp1Cdc20 levels rescue the TBZ sensitivity of dnt1Δ cells. (F) Elevated Slp1Cdc20 levels reverse the anaphase initiation defect upon SAC inactivation in dnt1Δ cells. (G) Schematic summary of data from (B-F). The balance of relative abundance between Slp1Cdc20 and checkpoint proteins governs the robustness of spindle checkpoint and thus mitotic exit rate. Lighter or darker shading depicts lower or higher levels of Slp1Cdc20 present in MCC or APC/C in dnt1Δ or slp1-overexpression (slp1) cells.

Artificially increased Slp1 abundance mitigates TBZ sensitivity of dnt1Δ cells

Based on the above results, we wondered whether the decreased levels of Slp1Cdc20 may be the major cause of the TBZ sensitivity and the defective anaphase initiation upon SAC inactivation in dnt1Δ cells. To test this possibility, we artificially increased Slp1Cdc20 abundance by expressing one or two extra copies of slp1 under its endogenous regulatory sequences (Fig 3C). Interestingly, two and three copies of slp1 restored abundance of Slp1Cdc20 in metaphase-arrested dnt1Δ cells to close to or slightly above the endogenous level in wild-type cells (Fig 3D). Consequently, sensitivity of dnt1Δ cells to TBZ was largely but not completely suppressed by excessive Slp1Cdc20 expression achieved by three copies of slp1 (Fig 3E). It should be noted that although the ectopic expression of leu1-32::slp1 restored Slp1Cdc20 in dnt1Δ cells almost to the same levels as in wild-type cells, it still could not rescue sensitivity of dnt1Δ cells to TBZ (Fig 3E). Nevertheless, increased Slp1Cdc20 abundance compromised the checkpoint activation in both wild-type and dnt1Δ cells, as revealed by lower percentages of cells with Cdc13-GFP at SPBs and metaphase-arrest upon cold-shock treatment, and the extent of failed checkpoint response was largely proportional to Slp1Cdc20 levels (Fig 3F and 3G). This result is consistent with previous reports in both fission and budding yeast that have underlined the importance of accurate relative abundance between checkpoint proteins and Cdc20/Slp1Cdc20, which sets an important determinant of checkpoint robustness [58, 59]. However, we noticed that the disappearance rate of Cdc13-GFP from SPBs was not accelerated in either wild-type or dnt1Δ background strains when Slp1Cdc20 abundance was artificially increased, regardless of the copy number of Slp1Cdc20 present (Fig 3F), indicating that the SAC silencing tempo was not altered by Slp1Cdc20 overexpression.

Together, these results suggested that the excessive Slp1 by three copies of slp1 antagonizes the negative effects of dnt1 deletion on TBZ sensitivity and lowered Slp1Cdc20 protein levels may only partly contribute to the TBZ sensitivity of dnt1Δ cells. The failure of complete rescue of the TBZ sensitivity of dnt1Δ cells by artificially increased Slp1Cdc20 abundance is possibly due to the retained and prolonged MCC-APC/C association, which still poses inhibition on APC/C activity.

Enhanced MCC-APC/C association and lowered Slp1Cdc20 abundance in dnt1Δ cells can be reversed by depletion of Apc15

It has been shown in both human and fission yeast cells that Apc15 mediates MCC binding to APC/C and is required for Cdc20/Slp1 autoubiquitylation and its turnover by APC/C [22,55,60,61]. Since our above data suggested that the TBZ sensitivity of dnt1Δ cells is likely caused by at least two aspects of defects in this mutant: one is the prolonged MCC-APC/C association, and the other is the lowered Slp1Cdc20 protein level, we reasoned that the absence of Apc15 may reverse the positive effect of deletion of dnt1 on MCC-APC/C association and its negative effect on Slp1Cdc20 levels.

We first investigated how the absence of apc15 affected the MCC-APC/C interaction in dnt1Δ cells. By immunoprecipitation of Apc4/Lid1-TAP, we found that the absence of Dnt1 enhanced MCC-APC/C interaction as more Mad2 and Mad3 were co-immunoprecipitated in dnt1Δ cells when compared to those in wild-type cells, whereas the deletion of apc15 abolished the MCC-APC/C interaction both in wild-type and dnt1Δ cells (Fig 4A). Quite unexpectedly, although the total cellular level of Slp1Cdc20 was significantly attenuated in dnt1Δ cells compared to that in wild-type cells (see Input in Fig 4A and 4B), we observed much more Slp1Cdc20 was co-immunoprecipitated by Apc4/Lid1-TAP but significantly reduced in Mad3-GFP immunocomplexes in dnt1Δ cells (Fig 4A and 4B). Interestingly, apc15Δ restored Slp1Cdc20 levels bound to MCC in dnt1Δ cells (Fig 4B). These observations seem to support the idea that the efficiency of Slp1Cdc20 being co-immunoprecipitated by APC/C subunit or MCC component differs significantly. Also, these data suggested that the increased APC/C-associated Slp1Cdc20 is likely “trapped” in the interface between MCC and APC/C and is unable to fulfill its function as the activator for APC/C.

Fig 4

apc15Δ reverses enhanced MCC-APC/C association and lowered Slp1Cdc20 abundance in dnt1Δ cells.

(A) apc15Δ abolishes the elevated binding of MCC to APC/C in dnt1Δ cells. The association of Mad2, Mad3 and Slp1Cdc20 to Apc4/Lid1 was assessed by immunoprecipitation of Apc4/Lid1-TAP in checkpoint-arrested cells as in Fig 2F. Note that more Mad2, Mad3 and Slp1Cdc20 was co-immunoprecipitated in dnt1Δ cells compared to wild-type cells, although the amount of Slp1Cdc20 was less abundant in dnt1Δ cells than that in wild-type cells. Results shown are the representative of three independent experiments. (B) apc15Δ restores the amount of Slp1Cdc20 bound to MCC in dnt1Δ cells. The assembly of MCC was assessed by immunoprecipitation of Mad3-GFP in checkpoint-arrested cells as in (A). Note that less Slp1Cdc20 was co-immunoprecipitated in dnt1Δ cells compared to wild-type cells. Results shown are the representative of three independent experiments. (C) apc15Δ restores the abundance of Slp1Cdc20 in dnt1Δ cells. Strains with indicated genotypes were grown and treated as in (A) to enrich checkpoint-arrested cells. Slp1Cdc20 levels were quantified with the relative ratio between Slp1Cdc20 and Cdc2 in wild-type strain set as 1.0. The experiments were repeated 3 times and the mean value for each sample was calculated as in Fig 3D. Error bars correspond to standard deviation. ***, p<0.001; **, p<0.01; n.s., not significant. (D) apc15Δ suppresses the delayed APC/C activation defect in dnt1Δ cells. Cells were synchronized by HU and then arrested at 18°C for 6 hours before being released at 30°C. The percentage of cells with Cdc13-GFP on SPBs was assessed at each time point as in Fig 2C. (E) Schematic summary of data from (A-D). White wavy lines depict the absence of Apc15. Lighter or darker shading depicts lower or higher levels of Slp1Cdc20 present in MCC or APC/C in mutant cells compared to wild-type cells.

Next, we examined whether depletion of Apc15 could rescue decreased Slp1Cdc20 abundance in dnt1Δ cells. As expected, we indeed observed that the absence of apc15 restored Slp1Cdc20 to a level that was even higher than that in wild-type cells (Fig 4C). This quantitative data suggested that the attenuated Slp1Cdc20 levels in dnt1Δ cells were most likely due to Apc15-faciltated degradation once Slp1Cdc20 is incorporated in MCC.

Furthermore, the absence of Apc15 also compromised the spindle assembly checkpoint response to disruption of spindles in dnt1Δ cells due to the loss of MCC-APC/C association, as the percentages of cells with Cdc13-GFP at SPBs were sharply reduced from over 90% to only about 70% in apc15Δ cells after nda3-mediated SAC activation (Fig 4D). These results are consistent with previous report showing that the relatively higher amount of Slp1Cdc20 can override the inhibitory effect of checkpoint proteins on APC/C activation [58]. Our results also suggested that Dnt1 is required for antagonizing the APC/C-mediated Slp1Cdc20 degradation and maintaining Slp1Cdc20 above its threshold level necessary for activating APC/C once the inhibitory signal from SAC is removed (Fig 4E).

Human CUEDC2 can partially rescue the TBZ sensitivity and spindle checkpoint inactivation defects of dnt1Δ cells

In humans, CUEDC2 mediates the release of APC/CCdc20 activity from Mad2 inhibition, and depletion of CUEDC2 causes a checkpoint-dependent delay of the metaphase-anaphase transition [27]. Since deletion of dnt1 in fission yeast also causes a checkpoint-dependent delay in anaphase entry, we wondered whether human CUEDC2 and fission yeast Dnt1 might share similar functions. To test this possibility, we ectopically expressed human CUEDC2 in fission yeast cells, and examined whether it could rescue the TBZ sensitivity and anaphase initiation defects upon SAC inactivation in dnt1Δ cells. Interestingly, the expression of nuclear localized CUEDC2 [i.e. CUEDC2 tagged with two copies of SV40 nuclear localization sequence (2×NLS)] slightly restored their growth at low concentrations of TBZ and efficiency of anaphase entry after SAC inactivation (S7 Fig). Cytoplasmic CUEDC2 did not have the same effect (S7B Fig), which was likely due to the fact that fission yeast undergoes “closed” mitosis, during which APC/C and MCC function only inside the nucleoplasm. On the contrary, similarly expressed human p31comet, another factor with verified function in timely spindle checkpoint silencing by promoting the release of Mad2 from MCC [32–35, 37], failed to exert a rescuing effect on TBZ sensitivity of dnt1Δ cells (S7B Fig). Interestingly, we found limited homology between N-terminal portion of Dnt1 and CUEDC2 (S8 Fig). These data raised a possibility that fission yeast Dnt1 might be a functional homologue of human CUEDC2 and they may share a similar function in releasing APC/CCdc20 from checkpoint inhibition during mitotic exit.

Dnt1 associates with APC/C upon SAC activation and during anaphase initiation

It has been previously shown that Cdk1-phosphorylated CUEDC2 directly binds to human Cdc20, and mediates the release of APC/CCdc20 activity from Mad2 inhibition [27]. To further dissect the possible mechanism that how Dnt1 promotes SAC inactivation, facilitates APC/C activation and maintains Slp1Cdc20 stability, we examined whether Dnt1 also similarly associated with Slp1Cdc20 or APC/C. By immunoprecipitation of sfGFP-Slp1 from SAC-activated cells arrested by nda3-KM311, we found limited amount of co-purified Dnt1 in addition to APC/C subunit Apc4/Lid1 and MCC component Mad2 (Fig 5A). Weak association between Dnt1 and APC/C was also detected in mitotically arrested but not asynchronized cultures by immunoprecipitation of Apc4/Lid1-TAP (Fig 5B and 5C). On the contrary, despite our intensive efforts, we were unable to detect the interaction between Mad2 or Mad3 and Dnt1 by co-immunoprecipitation (Fig 5D). These data suggested that very likely Dnt1 physically interacts more directly with APC/C but not MCC. Supporting this scenario, the interaction between sfGFP-Slp1 and Dnt1 was enhanced by depletion of Mad3 (Fig 5E), possibly because more APC/C assemblies devoid of MCC are available, thus Dnt1 gains better access to APC/C in mad3Δ cells. Unfortunately, we failed to detect the direct interaction between Dnt1 and APC/C subunits including Slp1Cdc20 by yeast two-hybrid analyses (S6 Fig). These data suggested that possibly Dnt1 physically interacts with APC/C through a “mediating” factor, which remains to be identified in the future studies.

Fig 5

Dnt1 associates with APC/C upon SAC activation and during anaphase initiation.

(A) Slp1Cdc20 co-immunoprecipitates Dnt1 in metaphase-arrested and mitosis-exiting cells. nda3-KM311 mad2-13myc strains simultaneously carrying either sfGFP-slp1 or dnt1-13myc or both were arrested in mitosis at 18°C and then released to 30°C, samples were collected at 0 and 40 min. Whole-cell extracts (input) were incubated with GFP-Trap beads and immunoprecipitated (IP) fractions were analyzed by immunoblotting. (B, C) Dnt1 can be co-immunoprecipitated by Apc4/Lid1 in metaphase-arrested but not asynchronous interphase cells. nda3-KM311 strains with indicated genotypes were either arrested at metaphase at 18°C or grown as asynchronous cultures at 30°C. Apc4/Lid1-GFP or Apc4/Lid1-HA was immunoprecipitated with GFP-Trap beads or anti-HA antibodies respectively and purified fractions were analyzed by immunoblotting. (D) Dnt1 cannot be co-immunoprecipitated by Mad2 and Mad3 in metaphase-arrested cells. nda3-KM311 strains with indicated genotypes were arrested at metaphase as in (B). Both Mad2-GFP and Mad3-GFP were immunoprecipitated with GFP-Trap beads and purified fractions were analyzed by immunoblotting. The around 120 kDa band corresponding to Dnt1-13myc was not detectable in co-immunoprecipitated sample (lane #3). Note that one strain carrying apc4/lid1-TAP but without dnt1-13myc served as a positive control, and anti-myc antibodies cross-reacted with Apc4/Lid1-TAP. (E) Mad3 imposes inhibitory effect on association between Slp1 and Dnt1. cdc25-22 strains expressing the indicated tagged proteins in the presence or absence of mad3 were first arrested at 36°C for 3.5 hours and then released to 25°C, samples were collected at 60 min after release. Immunoprecipitations were performed as in (A). In Fig 5A–5E, all asterisks indicate the unspecific band recognized by anti-myc antibodies, and all results shown are the representative of three independent experiments.

Presence of Dnt1 is beneficial to maintaining APC/C activity and cell survival upon spindle checkpoint activation

Our above data suggested that the absence of Dnt1 causes prolonged and enhanced SAC activation. We wondered whether this defective SAC silencing is beneficial to cells. Very intriguingly, we noticed that individually isolated nda3-KM311 dnt1Δ cells formed far fewer colonies on solid rich medium at the permissive temperature 30°C than nda3-KM311 dnt1 cells (47% vs. 68% viability) when they were treated at 18°C for 10 hours to activate SAC (Fig 6A). In contrast, the viability of dnt1Δ cells was not affected by this transient cold-shock (Fig 6A). This data indicated that Dnt1 is required to maintain cell viability specifically upon spindle stress, although it is largely dispensable for spindle checkpoint activation (S2 Fig).

Fig 6

Dnt1 involves in antagonizing SAC and maintaining cell survival upon spindle checkpoint activation.

(A) nda3-KM311 dnt1Δ cells lose viability after transient cold-shock. nda3-KM311 dnt1, nda3-KM311 dnt1Δ or dnt1Δ cells were first grown at 30°C in liquid cultures and then being shifted to 18°C for 10 hours. Individual cells (n ≥80 for each strain) were isolated using tetrad dissection manipulator and placed on solid rich media at the permissive temperature of 30°C (Left). The number of colonies formed before and after cold-treatment was quantified (Right). (B) Overexpressed Dnt1 antagonizes negative effect of Mad2 overexpression on loss-of-function APC/C mutants. Serial dilutions (10-fold) of the indicated strains were spotted on YE plates and incubated at the indicated temperatures. Overexpression of Mad2 and Dnt1 was achieved under nmt1 promoter (P) or adh1 promoter (P), respectively. Note that cut20-100 P-mad2 mutant was lethal and thus not included in the spot assay.

It has been previously shown that overexpression of spindle checkpoint protein Mad2 can activate SAC and block anaphase entry even in the absence of spindle defects [55,62], this is due to the fact that SAC proteins function as potent inhibitors of APC/C. Consistently, it is not surprising that fission yeast cells with temperature-sensitive mutations in the APC/C subunits were sensitive to elevated levels of Mad2 (P-mad2) [62] (Fig 6B). Given that Dnt1 is required to promote timely disassembly of MCC and cell survival, we examined whether elevated levels of Dnt1 can reverse the deleterious effects of overexpressed Mad2 on temperature-sensitive APC/C mutants. As expected, ectopic expression of dnt1+ under the control of adh1 promoter (P-dnt1) rescued the growth defects of tested APC/C mutants overexpressing Mad2 (Fig 6B). Most strikingly, higher levels of Dnt1 allowed the survival of cut20-100 P-mad2 mutant, which is lethal by its own (Figs 6B and S9).

Together, these results revealed that Dnt1 is required for maintaining cell viability, and also for antagonizing SAC thus resulting timely and efficient anaphase initiation and mitotic exit, especially when the SAC needs to be inactivated.

Discussion

The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit ubiquitin ligase that triggers the metaphase-to-anaphase transition in the cell cycle by targeting the substrates cyclin B and securin for destruction. APC/C activity toward its substrates requires its co-activator Cdc20. To ensure that cells enter mitosis and partition their duplicated genome with high accuracy, APC/CCdc20 activity must be tightly controlled. So far, besides Cdc20, several factors have also been identified in higher eukaryotes to regulate APC/CCdc20 activity, such as mitotic protein kinases Cdk1 and Polo-like kinase 1 (Plk1), which increase Cdc20 binding and APC/CCdc20 activity by phosphorylating Apc1, Apc3 and likely also other subunits [63-65], and the mitotic checkpoint complex (MCC), which functions as a specific inhibitor of Cdc20 and APC/CCdc20 activity mediated mainly by Mad3/BubR1 binding to two molecules of Cdc20 [19,20]. The above Cdc20-regulating components and mechanisms are largely or most likely conserved in fission yeast [22,66].

In this study, we have identified the fission yeast nucleolar protein Dnt1 as a novel positive regulator of Slp1Cdc20 protein level and APC/CCdc20 activity, especially when cells are recovered from arrest by activated spindle checkpoint. Although not tested, it is possible that our observed synthetic lethality between dnt1Δ and mutants with compromised kinetochores, peri-centromeric heterochromatin, cohesins, and microtubule-kinetochore attachment (S1 Fig) is due to low Slp1Cdc20 level and resulted prolonged activated SAC signaling. Actually, one previous study has underlined the importance of accurate relative abundance both within checkpoint proteins and between checkpoint proteins and the checkpoint target Slp1Cdc20 [58].

Our observations that deletion of dnt1 causes elevated and prolonged MCC-APC/C association and reduced abundance of Slp1 when cells are recovered from activated spindle checkpoint arrest could be explained by several different possibilities. The simplest of these would be that Dnt1 directly interacts with Slp1Cdc20 or other APC/C subunits to prevent the excessive and prolonged MCC binding to APC/C and consequently impede the degradation of MCC-bound Slp1Cdc20 by partially activated APC/C, thus to maintain Slp1 above its threshold level when SAC is active (Fig 7). We favor the idea that Dnt1 directly regulates the MCC-APC/C, although the possibility of its indirect regulation cannot be excluded. In humans, depletion of CUEDC2 causes a checkpoint-dependent delay of the metaphase-anaphase transition, and Cdk1-phosphorylated CUEDC2 binds to Cdc20 directly and mediates the release of APC/CCdc20 activity from Mad2 inhibition [27]. Since deletion of dnt1 in fission yeast also causes a checkpoint-dependent delay in anaphase entry and Dnt1 physically interacts with APC/C, it is thus plausible to assume that fission yeast Dnt1 might function similarly to human CUEDC2, though Dnt1 and CUEDC2 do not share high homology in their sequences (S8 Fig). However, we were unable to detect any interaction between Dnt1 and Slp1Cdc20 and other APC/C subunits by yeast two-hybrid analysis (S6 Fig), this is very distinct from human CUEDC2, which directly binds to Cdc20 [27]. It is fairly possible that Dnt1 interacts with APC/C through an unidentified “mediator” protein, this may explain why Dnt1 comes down only weakly with Slp1Cdc20 or Apc4/Lid1 in our immunoprecipitation assays (Fig 5). Therefore, we considered the fission yeast Dnt1 as a peripheral and phase-specific activator rather than an integral and permanent component or subunit of APC/C for two reasons. First, previous proteomics analyses of purified APC/C or potential Dnt1-interacting proteins did not identify Dnt1 or any APC/C subunits, respectively [44,67]. Second, we could detect the physical interaction between Dnt1 and Slp1Cdc20 or Apc4/Lid1 by immunoprecipitation only in metaphase-arrested and mitosis-exiting cells (Fig 5A and 5B), but not in cells from asynchronized cultures (Fig 5C), suggesting Dnt1 may facilitate APC/C activity in a cell cycle stage-specific and transient manner. This timely involvement of Dnt1 may aid a rapid and complete release of APC/C activity once the SAC signaling is satisfied and switched off. It is possible that one or more extra unrecognized factors might exist in higher eukaryotes to regulate APC/CCdc20 activity in a way similar to Dnt1 does. In future, it would be interesting to know whether Dnt1 indeed interacts indirectly with Slp1Cdc20 itself, and whether and how Dnt1 might antagonizes the autoubiquitylation of Slp1Cdc20.

Fig 7

Proposed model for positive regulation of the APC/C activity by Dnt1.

Upon SAC activation, APC/C-associated Dnt1 is likely involved in preventing the excessive and prolonged MCC binding to APC/C and thus impeding the degradation of MCC-bound Slp1Cdc20 by partially activated APC/C. The presence of Dnt1 possibly help to maintain Slp1Cdc20 protein (likely including both MCC-bound and free Slp1Cdc20) level and APC/CCdc20 activity. Lighter brown color of Slp1MCC depicts lower levels of Slp1Cdc20 present in MCC in dnt1Δ cells, which is likely caused by Apc15-faciliated autoubiquitylation and degradation. Darker green color of Slp1APC/C depicts higher levels of activation-incapable Slp1Cdc20 “trapped” in APC/C in dnt1Δ cells. In parallel, Dnt1 may also indirectly regulate the inhibitory action of MCC on APC/C (dotted arrows) through a yet unrecognized factor/mechanism (question mark).

Materials and methods

Fission yeast strains and genetic analyses

Standard media (either YE (yeast extract) rich medium or EMM (Edinburgh minimal medium) and culturing methods were used [68,69]. G418 disulfate (Sigma-Aldrich), hygromycin B (Sangon Biotech) or nourseothricin (clonNAT; Werner BioAgents) was used at a final concentration of 100 μg/ml and thiabendazole (TBZ) (Sigma-Aldrich) at 5–15 μg/ml in YE media. For serial dilution spot assays, 10-fold dilutions of a mid-log-phase culture were plated on the indicated media and grown for 3 to 5 days at indicated temperatures. To examine the possible synthetic lethality of double mutants, at least 20 complete tetrads were dissected after each genetic cross. Yeast strains containing Cdc13-mCherry, Mad2-13myc and Mad3-13myc were generated by a PCR-based module method [70], with the DNA sequence information obtained from PomBase (https://www.pombase.org/). To create strains with an extra copy of slp1 or dnt1 at lys1 locus, the slp1 genomic region from 1,504 bp 5’ to stop codon of the open reading frame or 1852 bp of the open reading frame of dnt1 was first cloned into the vector pUC119-P-MCS-hphMX6-lys1* with adh21 promoter (P) maintained or removed using the ‘T-type’ enzyme-free cloning method [71]. Similar procedures were employed to construct pUC119-P-MCS-hphMX6-lys1*-based plasmids carrying sequences corresponding to human p31comet or CUEDC2 (with plasmid or cDNA kindly provided by Hongtao Yu or Jiahuai Han respectively). Then, GFP or two tandem SV40 NLS or both sequences was introduced in front of dnt1, p31comet or CUEDC2 coding sequences by Quikgene method [72]. Finally, the resultant plasmids were linearized by ApaI and integrated into the lys1 locus, generating the strains lys1Δ::P-slp1-T::hphMX6, lys1Δ::P-slp1-T::hphMX6, lys1Δ::P-dnt1-T::hphMX6, lys1Δ::P-GFP-dnt1-T::hphMX6, lys1Δ::P-GFP-p31-T::hphMX6, lys1Δ::P-2×NLS-p31-T::hphMX6, lys1Δ::P-2×NLS-GFP-p31-T::hphMX6, lys1Δ::P-GFP-CUEDC2-T::hphMX6, lys1Δ::P-2×NLS-CUEDC2-T::hphMX6, and lys1Δ::P-2×NLS-GFP-CUEDC2-T::hphMX6. The other strain with an extra copy of slp1 at leu1 locus has been described previously [58]. Strains used in this study are listed in S1 Table.

Minichromosome loss assay

The minichromosome loss assay was performed in cells bearing the Ch16 (ade6-M216) minichromosome and ade6-M210 allele as previously described [54,73]. The rate of minichromosome loss per division was calculated by dividing the number of at least half red-sectored colonies grown on YE containing 12 mg/L adenine at 30°C by the number of total colonies (red colonies were excluded from the count).

Cell synchronization methods

For cdc25-22 strains, cells were grown at 25°C until mid-log phase and arrested at late G2 phase by shifting to 36°C for 3.5 hr and released at 25°C. For nda3-KM311 strains, cells were grown at 30°C to mid-log phase, synchronized at S phase by adding HU (Sangon Biotech) to a final concentration of 12 mM for 2 hr followed by a second dose of HU (6 mM final concentration) for 3.5 hr. HU was then washed out and cells were released at specific temperatures as required by subsequent experiments.

Spindle checkpoint activation assay

For metaphase arrest due to spindle checkpoint activation by disrupting mitotic spindles and abolishing kinetochore-microtubule attachment using the cold-sensitive nda3-KM311 mutation, cells synchronized by HU at 30°C were released to 18°C for up to 9 hr. For spindle checkpoint activation by the absence of tension generated by a temperature-sensitive mutation of a cohesin subunit, psc3-1T, cells were first arrested at S phase by HU at 25°C and then released to 37°C. For both methods, cells were withdrawn at certain time intervals and fixed with cold methanol and stained with DAPI (4’, 6-diamidino-2-phenylindole, Sigma-Aldrich). 200–300 cells were analyzed for each time point.

Spindle checkpoint silencing assay

For checkpoint silencing assay in the absence of microtubules, mid-log phase ark1-as3 cdc13-GFP nda3-KM311 cultures were first synchronized with HU at 30°C and then arrested in early mitosis by shifting to 18°C for 6 hr. 5μM ATP analog 1-NM-PP1 (Toronto Research Chemicals) was added to inactivate ark1-as3 and therefore spindle checkpoint. For checkpoint inactivation by shifting microtubule-depolymerized nda3-KM311 cells back to permissive temperature, cells were first synchronized with HU at 30°C and then arrested in early mitosis by shifting to 18°C for 6 hr, followed by incubation at 30°C to allow spindle reformation and therefore spindle checkpoint silencing. For both methods, cells were withdrawn at certain time intervals and fixed with cold methanol and stained with DAPI. 200–300 cells were analyzed for each time point. Each experiment was repeated at least three times.

Yeast two-hybrid assay

For yeast two-hybrid analysis, the Matchmaker system (Clontech) was used. Bait plasmids were constructed into pGBKT7 vector. Prey plasmids were constructed into pGADT7 vector. Bait and prey plasmids were co-transformed into the AH109 strain, and transformants were selected on double dropout medium (SD-Leu-Trp). The bait-prey interaction, which would activate the HIS3 and ADE2 reporter genes, was assessed by the growth on the triple (SD-Leu-Trp-His) or quadruple (SD-Leu-Trp-Ade-His) dropout media.

Immunoblotting, immunoprecipitation and antibodies

Western blot and immunoprecipitation experiments were performed essentially as previously described [44]. Proteins were immunoprecipitated by IgG Sepharose beads (GE Healthcare) (for Apc4/Lid1-TAP), GFP-Trap beads (ChromoTek) (for sfGFP-Slp1 and Apc4/Lid1-GFP) or anti-HA antibody-coupled protein A/G beads (for Apc4/Lid1-HA). When necessary, 300 mM instead of 150 mM NaCl was used in lysate buffer to remove unspecific binding of proteins to beads. The primary antibodies used for immunoblot analysis of cell lysates and immunoprecipitates were peroxidase-anti-peroxidase (PAP) soluble complex (Sigma-Aldrich), rabbit polyclonal anti-Myc (GeneScript), mouse monoclonal anti-GFP (Beijing Ray Antibody Biotech), rat monoclonal anti-HA (Roche), mouse monoclonal anti-Cdc13 antibodies (Novus Biologicals) or rabbit polyclonal anti-Slp1 (generated at Xiamen University antibody facility using recombinant N-terminal 290 amino acids region of Slp1 (6His-Slp1(1-290aa) as antigens, same as previously described [57]). Cdc2 was detected using rabbit polyclonal anti-PSTAIRE (sc-53, Santa Cruz Biotechnology) as loading controls. Secondary antibodies were anti-mouse or anti-rabbit HRP conjugates (Thermo Fisher Scientific) and were read out using chemiluminescence.

Fluorescence microscopy and live-cell imaging

GFP- and mCherry-fusion proteins (such as Cdc13-GFP, Cut2-GFP, Dnt1-GFP, CUEDC2-GFP, Cdc13-mCherry, and mCherry-Atb2) were observed in cells after fixation with cold methanol. Cells were washed in PBS and resuspended in PBS plus 1 μg/ml DAPI. Photomicrographs were obtained using a Nikon 80i fluorescence microscope coupled to a cooled CCD camera (Hamamatsu, ORCA-ER). Time-lapse imaging of live cells was performed at 30°C using a Perkin Elmer spinning-disk confocal microscope (UltraVIEW VoX) with a 100x NA 1.49 TIRF oil immersion objective (Nikon) coupled to a cooled CCD camera (9100–50 EMCCD; Hamamatsu Photonics) and spinning disk head (CSU-X1, Yokogawa). Image processing, analysis and spindle length measurement were carried out using Element software (Nikon), ImageJ software (National Institutes of Health) and Adobe Photoshop.

Statistical analysis

For quantitative analyses of each experiment, at least 200 cells were counted for each time point or sample, and each experiment was conducted at least three times. Error bars correspond to standard deviation (SD) throughout.

Summary of synthetic lethality between dnt1Δ and mutants with defective chromosome segregation.

(PDF)

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Dnt1 is dispensable for activating the SAC in the absence of kinetochore-microtubule attachment or tension.

(PDF)

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Dnt1 is required to efficiently silence the spindle checkpoint when Aurora B kinase is inhibited in the absence of microtubules.

(PDF)

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Dnt1 is required for timely degradation of securin (Cut2 in S. pombe) upon SAC inactivation.

(PDF)

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Dma1 does not affect the abundance of Slp1Cdc20 or restore Slp1Cdc20 protein level in dnt1Δ cells during anaphase after SAC inactivation.

(PDF)

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Dnt1 does not directly associate with APC/C subunits based on yeast two-hybrid assays.

(PDF)

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Human CUEDC2 partially rescues the TBZ sensitivity and spindle checkpoint inactivation defects of dnt1Δ cells.

(PDF)

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Sequence alignment of S. pombe Dnt1 and human CUEDC2.

(PDF)

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Elevated expression of Mad2 causes strong synthetic lethality in cdc20-100 mutant background.

(PDF)

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Original images of uncropped blots.

(PDF)

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Yeast strains used in this study.

(DOC)

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Raw numerical data.

(XLSX)

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22 Jun 2022

Dear Dr Jin,

Thank you very much for submitting your Research Article entitled 'Recovery from spindle checkpoint-mediated arrest requires a novel Dnt1-dependent APC/C activation mechanism' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by two independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a revised version. We cannot, of course, promise publication at that time.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This manuscript describes interesting observations showing that loss of function of the nucleolar protein Dnt1 causes a delay in anaphase inactivation of the mitotic checkpoint complex (MCC) and a consequent delay in activation of the anaphase promoting complex (APC). These effects seem to be mediated through the APC co-activator Slp1 (aka Cdc20 in other systems). The authors show reduced Slp1 levels and persistent anaphase association of Slp1 and the MCC with the APC in dnt1∆ mutant cells. While these results are interesting and in general convincing, the underlying mechanism remains unclear. The impact of the manuscript would be greatly enhanced if the authors could clearly show mechanistically whether Dnt1 is directly or indirectly involved in the phenomena described and what is the underlying mechanism.

In addition, the manuscript is confusing and difficult to follow what the authors think their experimental results mean as you read them. It would be very helpful to build their model in the results as the manuscript progresses. For example, they should describe clearly the rationale for experiments, what hypotheses are being tested and whether the results are consistent with the hypothesis. As is, it is not clear always clear how the results fit into an overall model for what is going on.

Main points.

1) The authors want to make the case that Dnt1 directly regulates the MCC-APC. However, Dnt1 is already proposed to have a numerous other functions as well as being a component of the nucleolus. I wonder if the effects they are seeing are indirect and represent a downstream consequence to other defects in these cells.

The main argument for a direct role for Dnt1 is in Figure 5 which seeks to show that Dnt1 associates with Slp1 specifically in metaphase and anaphase and not asynchronous cells. These experiments seem a bit preliminary and should be taken further. The association of Dnt1 with Slp1 is shown in checkpoint arrested (metaphase) nda3 (tubulin mutant) cells and in these cells 40 minutes after release from the arrest when they are in anaphase. The data from asynchronous cells is not shown and should be. The other way the Dnt1-Slp1 association is shown is in mitotic cells generated through release from a G2 arrest (cdc25-22). However the association is not observed in these cells unless mad3 is deleted. It is not clear why mad3∆ would increase the interaction of Dnt1 with Slp1. The authors state that their data suggests that “Dnt1 physically interacts more directly with APC/C but not MCC. Supporting this scenario, the interaction between sfGFP-Slp1 and Dnt1 was enhanced by depletion of Mad3 (Fig. 5B).” I do not understand why their data suggests this, since Mad3 associates with Slp1 both when it is bound to APC and when it is not bound to APC. Why not test whether Dnt1 comes down with the APC directly by Lid1/Apc4 IPs in mitosis. This would bolster the argument that it is associating with the APC and not the free MCC.

Extensive proteomics previous proteomics analysis on the APC has not identified Dnt1, so the observed association with Dnt1 is surprising. Also, why does Dnt1 not come down with Mad3 or Mad2, which are associated with Slp1 both on and off the APC. I wonder whether Dnt1 comes down weakly with Slp1 because it is a substrate.

2) I had a hard time understanding the logic and overall conclusions in the section titled: “Enhanced MCC-APC/C association and lowered Slp1Cdc20 abundance in dnt1∆ cells can be reversed by depletion of Apc15”. The overall conclusion is well stated at the end of the section. It would be helpful to state this at the beginning by saying something like: “One possibility for Dnt1 function is…… Several lines of evidence support this model…” This would give the reader some context to make the rationale and significance of the subsequent experiments more apparent. This same critique (and suggestion) could be applied for much of the manuscript.

3) In Figure 4C the authors state that they are looking at the amount of Slp1 associated with the MCC but not the APC by examining how much Slp1 comes down in Mad3 immunoprecipitations. But if you IP Mad3 you should get both pools of Mad3 associated with just the MCC and pools associated with the APC, so I do not understand the rationale for why Mad3 IPs only pull down Slp1 associated with the MCC and not the APC.

Minor Points.

1) Lines 217-219: States “Consistently, deletions of mad2+ or bub1+ completely abrogated this effect of dnt1∆ on delaying spindle checkpoint inactivation (Fig. 2I).” I do not think this makes sense because in these cells there is no spindle checkpoint in the first place, so you cannot look at the effect dnt1∆ on checkpoint inactivation. I think these sentences could be deleted.

2) Lines 280-282: “……whereas the deletion of apc15+ abolished the MCC-APC/C interaction both in wild-type and dnt1∆ cells (Fig. 4B).” This seems to be true for Mad2 and Slp1, but not Mad3. Is this significant and has it been reported previously?

3) In Figure 6, why do the authors think that dnt1∆ cells do not survive prolonged checkpoint arrest and release? If anything one might think that they would be better at surviving because they arrest better. It seems more likely that they do not decisively turn off the checkpoint and activate the APC in anaphase, leaving some securin and cyclin around and that interferes with anaphase chromosome segregation and mitotic exit.

Reviewer #2: In this manuscript the author has examined the role of dnt1 in the timely inactivation of spindle assembly checkpoint to efficiently initiate anaphase. These events are crucial for proper recovery of cells after APC mediated inhibition by spindle checkpoint proteins and mitotic exit. The author have shown that the Dnt1 inhibit prolonged association of mitotic checkpoint complex with APC/C during spindle assembly checkpoint inactivation. The genetic interaction studies and expression analysis revealed that the Dnt1 positively regulates the protein level of Slp1/Cdc20 protein.

Over all the manuscript is well written and observations are informative and very interesting, there are some clarifications that I would like to be addressed before publication.

Fig. 2F and G. The association of Mad2 with Lid1 in the absence of dnt1 persist longer than Mad3 association (Fig. F) but in graph (G) it is shown otherwise.

Fig. 2A why there are two different concentration of HU was used for synchronization?

Fig. 3A The negative genetic interaction was observed with APC subunits cut9, cut20 and cut23 mutants but not with nuc2 and apc15 mutants. Author could please explain the reason for the same.

Fig. 3A Why slp1-362 mutant is not growing properly even at 25oC,

Fig. 3B The nda3KM311 synchronization strategy was not properly mentioned in figure legend like how long the cells were incubated at low temperature before releasing them at 30oC.

Fig. 3D If author has tried to express the Slp1 under nmt1 promoter to suppress the TBZ sensitivity of dnt1Δ cells. Further what could be the reason slp1 expression at leu1 locus cannot suppress the TBZ sensitivity but expression at lys1 locus suppresses well.

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Reviewer #1: No: Underlying numerical data not provided.

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

12 Aug 2022

Submitted filename: renamed_93c87.doc

Click here for additional data file.

24 Aug 2022

Dear Dr Jin,

We are pleased to inform you that your manuscript entitled "Recovery from spindle checkpoint-mediated arrest requires a novel Dnt1-dependent APC/C activation mechanism" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have fully addressed my concerns.

Reviewer #2: The authors have addressed all of the specific comments raised by this reviewer satisfactorily. The revised manuscript is substantially improved and this reviewer strongly recommends the manuscript to be accepted.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly:

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-22-00538R1

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

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Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.

9 Sep 2022

PGENETICS-D-22-00538R1

Recovery from spindle checkpoint-mediated arrest requires a novel Dnt1-dependent APC/C activation mechanism

Dear Dr Jin,

We are pleased to inform you that your manuscript entitled "Recovery from spindle checkpoint-mediated arrest requires a novel Dnt1-dependent APC/C activation mechanism" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Anita Estes

PLOS Genetics

On behalf of:

The PLOS Genetics Team

Carlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdom

plosgenetics@plos.org | +44 (0) 1223-442823

plosgenetics.org | Twitter: @PLOSGenetics