Mde4 is phosphorylated during mitosis and interacts with the Cdc14 like phosphatase, Clp1
Previous studies found that clp1
Δ mutants show chromosome segregation defects, implying that Clp1 substrates regulate chromosome segregation[15
]. To identify such substrates, we purified protein complexes containing Clp1 and interacting proteins from mitotic cells using the substrate trapping mutant of Clp1, Clp1-C286S, which is catalytically inactive but binds more strongly to phosphorylated substrates than wild-type Clp1[15
]. Tandem affinity purification[17
] was used to isolate Clp1-C286S protein complexes from metaphase-arrested cells. Protein complexes were then analyzed by tandem mass spectrometry. Numerous proteins that copurified with Clp1 were identified (data not shown) including the S. pombe
monopolin proteins, Pcs1 and Mde4. Mde4 has twelve potential Cdk1 phosphorylation sites (SP/TP motifs), and five of them were identified as phosphorylated by mass spectrometry (). However, sequence coverage of Mde4 from the mass spectrometry analysis was only 30%, raising the possibility that the other 7 Cdk1 sites might be phosphorylated as well. Pcs1 has one potential Cdk1 site but it was not identified as phosphorylated in vivo
(data not shown). To confirm the apparent interaction between monopolin and Clp1 identified using mass spectrometry, we carried out co-immunoprecipitation and western blotting experiments. Mde4-GFP co-immunoprecipitated with the substrate-trapping mutant, Clp1-C286S-13Myc, but not with wild-type Clp1-13Myc (), suggesting that the interaction between Clp1-C286S and Mde4 is mediated by phosphorylation on Mde4. Because Clp1 dephosphorylates sites phosphorylated by Cdk1, whose activity peaks in early mitosis, we investigated whether Mde4 is phosphorylated during mitosis. In synchronous cultures, slower migrating forms of Mde4-13Myc appeared during early mitosis, decreased during late mitosis, and then disappeared after mitotic exit (). To confirm that the shift was due to phosphorylation, Mde4-13Myc isolated from metaphase-arrested cells was treated with recombinant Clp1 purified from E. coli.
Treatment with Clp1 eliminated the migration shift of Mde4-13Myc (), confirming that Mde4 is phosphorylated in vivo, and that Clp1 can dephosphorylate Mde4 in vitro. Moreover, Mde4 purified from E. coli
could be efficiently phosphorylated by Cdk1 isolated from metaphase arrested cells, showing that Mde4 can be phosphorylated by Cdk1 in vitro (). To examine the contribution of Clp1 to Mde4 dephosphorylation in vivo we synchronized wild-type and clp1
Δ cells in metaphase, then released them from the arrest and monitored Mde4 phosphorylation. Although Mde4 is more highly phosphorylated at the metaphase arrest point in clp1
Δ cells (Supplemental Figure S1A
), Mde4 still becomes dephosphorylated as these cells exit mitosis (Supplemental Figure S1B
). Thus although Mde4 specifically purifies in a complex with the substrate trapping allele of Clp1, and can be dephosphorylated by it in vitro, our data indicate that other phosphatases are able to effect the dephosphorylation in vivo in the absence of Clp1. The identification of these phosphatases will be of considerable interest, and will be the subject of future studies. Together these data show that Mde4 is phosphorylated in early mitosis, and then becomes dephosphorylated in anaphase.
Mde4 is phosphorylated during mitosis and interacts with Clp1
Clp1 promotes loading of monopolin onto the spindle during anaphase
To test how phosphorylation affects Mde4 function, we examined whether changes in Mde4 phosphorylation state correlated with changes in Mde4 localization. Previous results showed that in interphase, when Mde4 is dephosphorylated, the Mde4-Pcs1 complex localizes to the nucleolus and to the kinetochores, which cluster at the nuclear periphery next to the spindle pole bodies (SPBs) [6
]. As cells enter mitosis and Mde4 becomes phosphorylated, Mde4-Pcs1 leave the nucleolus but remain at the kinetochores [6
]. Additionally we found that in anaphase, when Mde4 becomes dephosphorylated, Mde4 and Pcs1 localize to both ends of the anaphase spindle (). Because Mde4 is more highly phosphorylated in clp1
Δ cells just prior to anaphase onset, we examined Pcs1 and Mde4 localization in c
Δ mutants as they progressed through mitosis. Pcs1-GFP localization was observed by time-lapse microscopy, using cells co-expressing the SPB marker Cdc11-GFP to monitor cell cycle progression (). In wild-type cells faint Pcs1-GFP spots, presumably corresponding to the kinetochores, could be observed between the two SPBs in early mitosis. At anaphase onset Pcs1-GFP localized to the spindle (, red arrow) and then to both ends of the spindle at late anaphase. In c
Δ cells, Pcs1-GFP spots at putative kinetochores were observed as in wild-type cells, however Pcs1-GFP was only observed faintly at both ends of the spindle at late anaphase (). As with Pcs1-GFP, Mde4-GFP localized faintly on the spindle in late, but not early, anaphase in c
Δ cells (). The reduced spindle localization of Mde4 and Pcs1 in c
Δ cells was not due to reduced protein levels since both proteins were present at or above wild-type levels in c
Δ cells (Figure S1A
, and data not shown). In interphase, localization of Mde4 and Pcs1 to the nucleolus and kinetochores was not affected in c
Δ cells (, and data not shown). These results suggest that dephosphorylation of Mde4 may be important for monopolin localization to the spindle. The ability of monopolin to localize to late anaphase spindles in c
Δ mutants is consistent with our results showing that other phosphatases besides Clp1 can promote Mde4 dephosphorylation.
Clp1 promotes loading of the monopolin onto the spindle during anaphase
Loss of monopolin causes defects in microtubule attachment to kinetochores and activates the spindle checkpoint
To test whether spindle localization of monopolin is important for spindle function, we examined spindle dynamics in monopolin mutants. Fission yeast spindle elongation can be divided into three distinct phases [18
]. During phase I, which begins when cells enter mitosis, the spindle forms and grows to span the nucleus at a length of 2.5~3µm. In phase II (metaphase/anaphase A), the spindle maintains the 2.5~3µm length, and then elongates during phase III, which corresponds to anaphase B. Mitotic spindles were observed by time-lapse fluorescent microscopy in wild-type, mde4
Δ, and pcs1
Δ cells expressing GFP-α-tubulin. A comparison of the kinetics of spindle elongation in mde4
Δ, and wild-type cells revealed that cells lacking monopolin spend significantly more time in phase II (). Wild-type cells stayed less than 20 min in phase II, but 8 of 14 mde4
Δ cells, and 6 of 8 pcs1
Δ cells showed a prolonged phase II suggesting a delay in metaphase. A metaphase delay is often observed in cells when microtubule attachments to kinetochores are defective, which triggers the spindle checkpoint to inhibit anaphase onset. Observation of asynchronous wild-type and mde4
Δ cells showed that an elevated percent of mde4
Δ cells displayed spindle checkpoint activation as judged by the presence of Mad2 at kinetochores (). We tested for genetic interactions between the mde4
Δ mutant and the spindle checkpoint mutants, mad1
Δ and bub1
Δ. The mde4
Δ mutation was synthetic lethal with bub1
Δ since no double mutants were recovered. Double mutants between mde4
Δ and mad1
Δ or mad3
Δ showed strong synthetic growth defects (), consistent with the notion that mde4
Δ cells have defects in attachment of microtubules to kinetochores, which trigger spindle checkpoint dependent delays.
Monopolin mutants have delays in anaphase onset and show occasional spindle collapse
We found another difference in spindle morphology in monopolin mutants. Spindle lengths at the phase II/III transition in mde4
Δ and pcs1
Δ mutants were longer, 3.52 ± 0.15 µm and 3.47 ± 0.23 µm respectively, than that of wild-type cells, 3.07 ± 0.23 µm (). Elongated phase II spindles have also been observed in mutants defective in either microtubule attachment to kinetochores [21
], or cohesion between sister chromatids [22
], which may suggest that poorly attached kinetochores or reduced cohesion in monopolin mutants causes an imbalance of pushing and pulling forces in the spindle leading a somewhat elongated metaphase spindle. Previous results showed that monopolin is involved in preventing merotelic attachments [6
], which are not thought to be monitored by the spindle checkpoint. However our results suggest an additional role for monopolin in attachment of microtubules to kinetochores or cohesion, which is monitored by the spindle checkpoint.
Monopolin may stabilize anaphase spindle microtubules to prevent chromosome co-segregation
The analysis of spindle elongation in mde4
Δ cells revealed a function for monopolin in promoting anaphase spindle stability. In 6.4% (3/47) of mde4
Δ cells the spindles appeared to break and/or collapse (, Figure S2A
, and Movie S1
, Movie S2
, and Movie S3
). Consistent with this, we found that about 8.5 % of septated mde4
Δ cells showed chromosome co-segregation, in which one daughter cell inherited all of the chromosomes (). This phenotype likely arises because the nuclear envelope does not break down during mitosis in S. pombe.
As a consequence of this, when the spindle breaks prematurely the chromosomes collapse back into a single mass inside the nuclear envelope. Similar results have been observed when the mitotic spindle was cut using laser microsurgery [19
]. In those studies, when the spindle was cut near the middle, the mitotic spindle completely collapsed. However, when the mitotic spindle was cut near one SPB, the spindle continued to elongate unidirectionally, with the end of the spindle lacking an SPB pushing out a finger of nuclear envelope as it elongated. Once the broken end of the spindle reached the end of the cell, it pushed against the cell tip causing the other spindle pole to move to the opposite end of the cell. Of the 3 cells that showed spindle elongation defects, 2 of them showed spindle breakage and collapse (, and Movie S1
and Movie S2
), and one showed unidirectional spindle elongation suggesting that the spindle may have broken near one pole (Movie S3
and Figure S2A
). Another case of apparent spindle breakage followed by chromosome co-segregation was observed by time-lapse analysis of mde4
Δ cells expressing histone H3-GFP (Figure S2B
). Furthermore, thin protrusions of nuclear envelope possibly caused by broken spindle ends were also observed in some mde4
Δ but not wild-type cells (Figure S2C
). Taken together, our data suggest that monopolin may have a novel function in stabilization of anaphase spindles.
Characterization of non-phosphorylatable and phospho-mimetic mutants of Mde4
To more clearly define the role of phosphorylation of Mde4 in regulating the monopolin complex, we constructed non phosphorylatable and phospho-mimetic mutants. Since incomplete sequence coverage of Mde4 in our mass spectrometry did not allow us to identify all in vivo
Cdk1 phosphorylation sites on Mde4, we mutated all twelve of the Cdk1 consensus sites to alanine (S/T to A) to prevent phosphorylation or to acidic residues (S to D and T to E) to create a phospho-mimetic mutant. We named them mde4-12A
respectively, and constructed strains where the endogenous mde4+
locus was replaced by mde4-12A
. As expected, the Mde4-12A mutant protein was no longer hyperphosphorylated in metaphase-arrested cells (Figure S3A
), and Mde4-12A was not phosphorylated by Cdk1 in vitro (Figure S3B
), indicating that the major sites of mitotic phosphorylation had been eliminated. Additionally, Mde4-12A did not interact with Clp1-C286S (Figure S3C
), showing that phosphorylation is required for interaction with the Clp1-C286S substrate trapping allele. We do not think that the 12A and 12D mutations grossly perturbed the Mde4 protein structure because they did not affect Mde4-GFP localization to kinetochores and the nucleolus in interphase cells (Figure S3D
). Additionally, kinetochore and nucleolar localization of Pcs1-GFP was abolished in the absence of Mde4 (Figure S3E
), but was retained in mde4-12A
mutants (Figure S3E
) suggesting that the Cdk1 site mutations do not disrupt formation of the monopolin complex.
Phosphorylation of Mde4 is required to prevent monopolin localization to the prometaphase/metaphase spindle
Unlike wild-type Mde4, Mde4-12A-GFP was often observed on the spindle in pre-anaphase cells (data not shown), suggesting that it may load onto the spindle prematurely. To examine this in greater detail, we arrested cells at metaphase by overexpressing the spindle assembly checkpoint protein Mad2, which prevents cyclin B and securin destruction by suppressing the activity of the APC/C-Cdc20 complex [24
]. Under these conditions, Mde4-GFP and Mde4-12D-GFP were seen as several faint dots between the two spindle pole bodies, which were labeled with Sid4-mRFP (). These dots co-localized with a kinetochore protein, Nuf2-mRFP (). In contrast to Mde4-GFP, Mde4-12A-GFP localized to the metaphase spindle and could not be detected at kinetochores (, yellow arrow). Mde4-12A localized to the metaphase spindle in 97% (n=115) of metaphase cells, in contrast to Mde4 (n=126) and Mde4-12D (n=103), which did not localize on the spindle in metaphase cells (). To examine how the mde4-12A
mutation affects Pcs1 localization, we counted cells showing spindle localization of Pcs1-GFP in asynchronous mde4+
cells and classified them into mononucleate (early mitosis) and binucleate cells (anaphase). In wild-type cells, Pcs1-GFP localization on the spindle was observed almost exclusively in anaphase cells (307 of 308). In mde4-12A
cells Pcs1-GFP localized normally to anaphase cells, but was also observed in pre-anaphase cell; specifically, 48% (n=350) of cells showing Pcs1-GFP localization to the spindle were mononucleates (). Thus, in each assay the non-phosphorylatable Mde4-12A mutant localizes the monopolin complex inappropriately to prometaphase/metaphase spindle, implying that Cdk1 phosphorylation acts to prevent premature localization of Mde4 to the mitotic spindle.
Mde4-12A prematurely localizes to the spindle instead of kinetochores before anaphase onset and displays lagging chromosomes in anaphase
Localization of monopolin at kinetochores has been proposed to prevent merotelic attachments, which cause lagging chromosomes [6
]. We observed that 22% of mde4-12A
cells (n=304) showed lagging chromosomes in anaphase, which is comparable to the 31% observed in mde4
Δ cells (n=327) (). In contrast, less than 1% of mde4-12D
cells (n=311) in anaphase showed lagging chromosomes (). Our data shows that Cdk1 phosphorylation on Mde4 is important for its role in preventing merotelic attachment of kinetochores to spindle microtubules. One explanation for this could be that Cdk1 phosphorylation promotes kinetochore localization of Mde4 by inhibiting premature localization of Mde4 to microtubules in prometaphase/metaphase.
Mde4 Dephosphorylation promotes localization of monopolin to anaphase spindles
We used the mde4-12A and mde4 12D mutants to explore the role of Mde4 dephosphorylation in monopolin localization to the anaphase spindle (). Mde4-GFP and Mde4-12A-GFP localized similarly to the spindle in early and late anaphase. In contrast, Mde4-12D-GFP showed a diffuse nuclear signal in anaphase, with less than 10% of cells showing faint spindle localization. Pcs1-GFP localized in mde4+, mde4-12A, and mde4-12D cells the same as each Mde4 mutant protein ( lower panel), consistent with the notion that the phosphorylation status of Mde4 regulates localization of the monopolin complex to the spindle. The poor localization of Mde4-12D to the spindle is similar to that observed for wild-type Mde4 in early anaphase clp1Δ cells, presumably because Mde4 is not dephosphorylated as rapidly in clp1Δ cells. If this is the case, then disruption of Mde4 phosphorylation should rescue the Mde4 spindle localization defect in clp1Δ cells. This proved to be the case, since Mde4-12A localized strongly to anaphase spindles in clp1Δ cells (). Additionally, expression of Mde4-12A was sufficient to restore Pcs1 association with the mitotic spindle (). Together these results show that Cdk1 phosphorylation on Mde4 must be removed to allow the monopolin complex to localize to anaphase spindles.
Mde4-12D localizes poorly to anaphase spindles
Both mde4-12A and mde4-12D cells display distinct defects in spindle elongation
To better characterize spindle dynamics in mde4-12A and mde4-12D mutants, spindle elongation was observed by time-lapse microscopy in these cells. Spindle elongation in mde4-12A cells resembled mde4Δ cells (compare with ), in that 50% (10 of 20) mde4-12A cells remained longer in phase II than mde4+ cells. However unlike mde4Δ cells, we did not observe spindle collapse or breakage in mde4-12A cells. The extended phase II in mde4-12A cells suggests that, like mde4Δ cells, they have microtubule attachment defects that trigger a spindle assembly checkpoint-dependent delay. To test this idea, we crossed mde4-12A to various spindle checkpoint mutants. The mde4-12A mutant was synthetically lethal with the mad1Δ, mad2A, mad3Δ and bub1Δ checkpoint mutants (), suggesting that the extended duration at phase II in mde4-12A cells is due to the activation of the spindle checkpoint. Notably, this interaction is even stronger than that observed for mde4Δ, since mde4Δ was only lethal with bub1Δ. Additionally, mde4-12A cells displayed significantly longer spindle lengths at phase II/III transition (3.78±0.12µm) than both wild-type and mde4Δ cells (). These results suggest that mde4-12A mutants may have greater defects in microtubule attachment to kinetochores than mde4Δ cells. This could be explained if the defects in mde4-12A cells are due not just to loss of Mde4 from the kinetochores in metaphase, but also to premature localization of Mde4 to the spindle.
Both mde4-12A and mde4-12D cells display distinct defects in spindle elongation
Overall mde4-12D cells showed spindle behavior similar to wild-type cells but most cells (14 of 16) displayed slightly shorter phase II ( and ). Spindle lengths at the phase II/III transition in mde4-12D cells were 2.97 ± 0.09 µm, which is similar to that of wild-type cells (), and mde4-12D cells did not show negative genetic interactions with the spindle checkpoint mutants, mad1Δ, mad2Δ, mad3Δ and bub1Δ (), suggesting that microtubule attachment in mde4-12D cells is normal. A small fraction of mde4-12D cells exhibited spindle collapse followed by recovery (). Furthermore, 2.3% of mde4-12D cells (13 of 561) showed chromosome co-segregation (). While this represents a minority of mde4-12D cells, this was not observed in an equivalent number of wild-type cells (). These data are consistent with the view that reduced levels of Mde4 on the spindle may compromise its stability. Unexpectedly, we also found that mde4-12D cells display a reduced rate of spindle elongation in early anaphase B (). We measured the spindle elongation rate during early (3.5 to 6 µm length of the spindle) and late (6 to 10 µm length of the spindle) phase III. Although in wild-type cells the rate of spindle elongation (~0.65 µm/min) was the same in early and late phase III, in mde4-12D cells the rate of spindle elongation was slower during early phase III (~0.40 µm/min) but was similar to wild-type cells in late phase III (~0.74 µm/min). Taken together, our data suggests that dephosphorylation of Mde4 at Cdk1 sites triggers spindle localization of monopolin, which promotes proper spindle elongation in early anaphase and enhances spindle stability.