Sister centromere fusion during meiosis I depends on maintaining cohesins and destabilizing microtubule attachments

Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. We demonstrate that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, we propose two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments.


Introduction
The necessity of sister kinetochores to co-orient toward the same pole for co-segregation at anaphase I differentiates the first meiotic division from the second division. A meiosis-specific mechanism exists that ensures sister chromatid co-segregation by rearranging sister kinetochores, aligning them next to each other and facilitating microtubule attachments to the same pole [1,2]. We refer to this process as co-orientation, in contrast to mono-orientation, when homologous kinetochores orient to the same pole. Given the importance of co-orientation in meiosis the mechanism underlying this process is still poorly understood, maybe because many of the essential proteins are not conserved across phyla.
Most studies of co-orientation have focused on how fusion of the centromeres and kinetochores is established. In budding yeast, centromere fusion occurs independently of cohesins: Spo13 and the Polo kinase homolog Cdc5 recruit a meiosis-specific protein complex, monopolin (Csm1, Lrs4, Mam1, CK1) to the kinetochore [3][4][5]. Lrs4 and Csm1 form a V-shaped structure that interacts with the N-terminal domain of Dsn1 in the Mis12 complex to fuse sister kinetochores [6,7]. While the monopolin complex is not widely conserved, cohesin-independent mechanisms may exist in other organisms. A bridge between the kinetochore proteins MIS12 and NDC80 is required for co-orientation in maize [8]. In contrast, cohesins are required for co-orientation in several organisms. The meiosis-specific cohesin Rec8 is indispensable for sister centromere fusion in fission yeast [9] and Arabidopsis [10,11]. Cohesin is localized to the core-centromere in fission yeast [12] and mice [13]. In Drosophila melanogaster oocytes, we and others have shown that cohesins (SMC1/SMC3/SOLO/SUNN) establish cohesion in meiotic S-phase and show an enrichment that colocalizes with centromere protein CID/CENP-A [14][15][16][17]. Like fission yeast and mouse, Drosophila may require high concentrations of cohesins to fuse sister centromeres together for co-orientation during meiosis.
In mice, a novel kinetochore protein, Meikin, recruits Plk1 to protect Rec8 at centromeres [13]. Although poorly conserved, Meikin is proposed to be a functional homolog of Spo13 in budding yeast and Moa1 in fission yeast. They all contain Polo-box domains that recruit Polo kinase to centromeres [13]. Loss of Polo in both fission yeast (Plo1) and mice results in kinetochore separation [13,18], suggesting a conserved role for Polo in co-orientation. In fission yeast, Moa1-Plo1 phosphorylates Spc7 (KNL1) to recruit Bub1 and Sgo1 for the protection of centromere cohesion in meiosis I [18,19]. These results suggest the mechanism for maintaining sister centromere fusion involves kinetochore proteins recruiting proteins that protect cohesion. However, how centromere cohesion is established prior to metaphase I, and how sister centromere fusion is released during meiosis II, still needs to be investigated.
We previously found that depletion of the kinetochore protein SPC105R (KNL1) in Drosophila oocytes results in separated centromeres at metaphase I, suggesting a defect in sister centromere fusion [20]. Thus, Drosophila SPC105R and fission yeast Spc7 may have conserved functions in co-orientation [18]. We have identified a second Drosophila protein required for sister-centromere fusion, Protein Phosphatase 1 isoform 87B (PP1-87B). However, sister centromere separation in SPC105R and PP1-87B depleted Drosophila oocytes occurs by different mechanisms, the former is Separase dependent and the latter is Separase independent. Based on these results, we propose a model for the establishment, protection and release of co-orientation. Sister centromere fusion necessary for co-orientation is established through cohesins that are protected by SPC105R. Subsequently, PP1-87B maintains co-orientation in a cohesinindependent manner by antagonizing stable kinetochore-microtubule (KT-MT) interactions. The implication is that the release of co-orientation during meiosis II is cohesin-independent and MT dependent. We also found a surprising interaction between PP1-87B and C(3)G, the transverse element of the synaptonemal complex (SC), in regulating sister centromere separation. Overall, our results suggest a new mechanism where KT-MT interactions and centromeric SC regulate sister kinetochore co-orientation during female meiosis.

PP1-87B is required for chromosome organization and sister centromere fusion in meiosis I
Drosophila has three homologs of the alpha type of mammalian Protein Phosphatase1 (PP1α/ γ) genes, Pp1-87B, Pp1-96A and Pp1-13C [21]. We focused our studies on the Pp1-87B isoform because it is the only essential gene, is highly expressed during oogenesis, and contributes 80% of PP1 activity during development [21,22]. As Pp1-87B mutations are lethal, tissue-specific expression of an shRNA targeting Pp1-87B was used to define its role in oocytes (see Methods) [23]. The ubiquitous expression of an shRNA for PP1-87B using tubP-GAL4-LL7 resulted in lethality, suggesting the protein had been depleted. When PP1-87B was depleted in oocytes using mata4-GAL-VP16 (to be referred to as Pp1-87B RNAi oocytes), we observed two phenotypes. The first was disorganization of the metaphase I chromosomes. In wild-type Drosophila oocytes, meiosis arrests at metaphase I with the chromosomes clustered into a single chromatin mass at the center of the spindle (Fig 1A). In 62% of Pp1-87B RNAi oocytes, the single chromosome mass was separated into multiple groups of chromosomes (Fig 1A and 1B). The second phenotype observed in Pp1-87B RNAi oocytes was precocious separation of sister centromeres, as determined by counting the number of centromere protein CENP-C or CID (CENP-A) foci (see Methods) [24]. In wild-type oocytes, we observed an average of 7.3 centromere foci, consistent with the eight expected from four bivalent chromosomes at metaphase I (Fig 1A and 1C). However, in Pp1-87B RNAi oocytes we observed a significantly higher number of foci (mean = 12.7). This suggests a defect in sister centromere fusion results in their premature separation during metaphase I.
To determine whether the separated chromosome mass and centromere separation phenotypes in Pp1-87B RNAi oocytes is caused by a general loss of cohesion, we used heterochromatic FISH probes directed to the pericentromeric regions of each autosome to mark the homologs. In wild type, two FISH foci are typically observed per homologous chromosome pair in metaphase I because of cohesion between sister chromatids (Fig 1D). To determine if pericentromeric cohesion in Pp1-87B RNAi oocytes was affected, we analyzed the number of heterochromatin FISH signals from the dodeca satellite, the most punctate and therefore quantifiable heterochromatic FISH probes. In ord mutant oocytes that lack all cohesion, the oocytes had a significantly higher number of dodeca foci (mean = 4.8) compared to wild type two FISH foci in separate chromosome masses. We observed that while 62% of Pp1-87B RNAi oocytes (n = 50) had a separated chromosome mass, only 8.5% of the homologs had separated (n = 130). These results suggest that arm cohesion is usually retained when PP1-87B is depleted. Hence, the separated chromosome mass phenotype in Pp1-87B RNAi oocytes is due to intact bivalents failing to organize correctly at the center of the spindle.
Based on these FISH results, PP1-87B is only required for maintaining sister centromere cohesion but is dispensable for cohesion of the pericentromeric regions and the chromosome arms in oocytes. To refer to this specific type of cohesion, we will use the term sister centromere fusion. We also observed two defects associated with the defect in sister centromere fusion and a lack of co-orientation in Pp1-87B RNAi oocytes. First, the FISH experiments can detect errors in homologs bi-orientation, defined as when pairs of homologous centromeres are separated towards opposite poles (Fig 1D). In Pp1-87B RNAi oocytes, 5.3% of the homologs were mono-oriented, defined as when pairs of homologous centromeres are have moved towards the same pole (n = 130 vs. n wt = 111, p = 0.016). These results support the conclusion that the sister centromere fusion defect in Pp1-87B RNAi oocytes causes problem for homologous chromosomes to bi-orient.
Second, when the sister centromeres that precociously separate dring meiosis I in mouse and yeast, chiasmata can still direct bi-orientation of these homologs, suppressing the consequences of co-orientation defects [9,13,25]. Therefore, we used a crossover defective mutant, mei-P22 [26], to generate univalents, and knocked down Pp1-87B in these oocytes. If the precocious sister centromere separation causes a co-orientation defect, we would expect the univalents in mei-P22, Pp1-87B RNAi oocytes can become bi-oriented. Indeed, we observed that 20% of mei-P22, Pp1-87B RNAi oocytes had sister chromatids bi-oriented (n = 15, Fig 1F). These results suggest that PP1-87B is required for sister centromere fusion to facilitate co-orientation in metaphase I of oocytes.

Co-orientation in Drosophila oocytes requires both cohesin-dependent and cohesin-independent pathways
Both cohesin-dependent and -independent mechanisms of sister centromere fusion have been described. Therefore, we investigated whether loss of sister centromere fusion depends on cohesin release. In addition to PP1-87B, the kinetochore protein SPC105R was also tested because it is the only other protein known to be required for sister centromere fusion in Drosophila oocytes [27]. To investigate if cohesin is involved in sister centromere fusion, we tested if sister centromere separation in Pp1-87B-and Spc105R-RNAi oocytes depends on known cohesin removal mechanisms by depleting two negative-regulators of cohesin, Wings Apartlike (wapl) and Separase (sse). If losing a factor required for cohesin removal rescued the sister centromere separation in Pp1-87B or Spc105R RNAi oocytes, it would suggest the Drosophila sister centromere fusion depends on cohesin.
Upon co-expression of wapl shRNA with either Pp1-87B or Spc105R shRNA, the centromeres remained separated (Fig 2A and 2B). While WAPL could be required for cohesion release at anaphase I, these results suggest WAPL is not required for the meiosis I sister centromere separation caused by depletion of PP1-87B or SPC105R. However, the centromeres in wapl, Pp1-87B RNAi oocytes became thread-like instead of punctate (Fig 2A), leading to additional centromere foci when quantified (Fig 2B). The thread-like centromere phenotype suggests that chromosome structure is affected in wapl, Pp1-87B RNAi oocytes, consistent with previous studies that concluded WAPL was involved in regulating chromosome structure [28,29].
The separated centromere phenotype was rescued in sse, Spc105R RNAi oocytes (Fig 2A  and 2C; mean = 8.1), suggesting that centromere separation in Spc105R RNAi oocytes depends on the loss of cohesins. This is a surprising result because it suggests that Separase is active  (12), Spc105R RNAi (26), wapl RNAi + Spc105R RNAi (21), Pp1-87B RNAi (27), wapl RNAi + Pp1-87B RNAi (13), sse RNAi (11), sse RNAi + Spc105R RNAi (36), sse RNAi + Pp1-87B RNAi (18). during meiotic metaphase I [30]. If Separase is active, these results could be explained if SPC105R recruits proteins that protect cohesins from Separase. To test the hypothesis that SPC105R protects cohesins from Separase, we examined the localization of MEI-S332/SGO, which is required to maintain cohesion during meiosis in several organisms [31]. Drosophila orthologue MEI-S332 localizes to centromere and peri-centromeric regions in wild-type meiosis I oocytes, as shown by colocalization and substantial non-overlap distribution with the core centromere (S1 Fig.). While present during meiosis I and useful as a marker for cohesion protection, MEI-S332 only shows defects during meiosis II [32,33], possibly due to redundancy with another factor during meiosis I [34,35]. Consistent with our hypothesis, MEI-S332 localization was almost abolished in Spc105R RNAi oocytes (Fig 3A and 3B). While we cannot rule out non-cohesive functions for Separase, the most likely interpretation is that SPC105R is required to recruit proteins that protect cohesins from Separase.
On the other hand, different from the result of sse, Spc105R RNAi, the separated centromere phenotype was not rescued in sse, Pp1-87B RNAi oocytes (Fig 2A and 2C; mean = 13.4). Consistent with cohesin-independence of these phenotypes, the localization of MEI-S332 in Pp1-87B RNAi oocytes was not reduced, and in fact, the volume was increased relative to wild-type ( Fig 3A and 3B). Aurora B is required for MEI-S332 localization [36], and although the mechanism is not well understood in Drosophila, our results suggest MEI-S332 localization is promoted by Aurora B but constrained by PP1-87B. These results indicate that sister centromere fusion in Drosophila oocytes is regulated through two different mechanisms: the SPC105R pathway that is sensitive to Separase, and the PP1-87B pathway that is Separase independent.

Separase-independent loss of sister centromere fusion depends on microtubule dynamics
Because the Pp1-87B RNAi phenotype was not suppressed by loss of Separase, we investigated cohesin-independent mechanisms for how PP1-87B regulates sister centromere fusion. A critical initial observation was that the spindle volume of Pp1-87B RNAi oocytes was larger than wild type ( Fig 4A). In addition, PP1-87B was found to localize to the oocyte meiotic spindle (S2 Fig.). Based on these observations, we tested the hypothesis that PP1-87B regulates microtubules dynamics by co-depleting proteins known to regulate MT dynamics and KT attachments.
Aurora B kinase activity is required for spindle assembly in Drosophila oocytes [37] and can be antagonized by PP1 in other systems [38]. Furthermore, they have opposite phenotypes: both the chromosome mass and sister centromeres precociously separate in Pp1-87B RNAi oocytes but remain together in Aurora B-depleted oocytes [37]. Therefore, we tested whether Aurora B is required for both the chromosome mass and centromere separation phenotypes of Pp1-87B RNAi oocytes. Treatment of metaphase I oocytes (i.e. those that have assembled a spindle) with the Aurora B inhibitor, Binucleine 2 (BN2) [39], caused loss of the spindle (65%, n = 29, Fig 4B and 4C), consistent with previous findings that Aurora B is required for spindle assembly [37]. Interestingly, Pp1-87B RNAi oocytes showed partial resistance to BN2 treatment; only 9% had lost the spindle and 50% of oocytes had residual MT around the chromosome mass (n = 22, Fig 4B and 4C). Regardless of these residual MTs, the increased number of centromere foci in Pp1-87B RNAi oocytes (mean = 13.0) was rescued by BN2 treatment to a level ( Fig 4D and 4E, mean = 7.4) similar to wild-type controls (Fig 4D and 4E, mean = 7.7). Similarly, the increased frequency of chromosome mass separation in Pp1-87B RNAi oocytes was rescued by BN2 treatment (Fig 4F and 4G). In contrast, centromere separation was not rescued by BN2 treatment of Spc105R RNAi oocytes (Fig 4B and 4C, mean = 11.3). These results are concordant with the effects of sse RNAi on the Spc105R and Pp1-87B RNAi phenotypes and support the conclusion that the maintenance of centromere fusion may occur by at least 2 mechanisms.
Suppression of Pp1-87B RNAi oocyte phenotypes by BN2 treatment could have been due to loss of Aurora B activity, or loss of the spindle microtubules. To distinguish between these two possibilities, we treated Pp1-87B RNAi oocytes with Paclitaxel to stabilize the spindle prior to BN2 treatment of the oocytes. Although these oocytes successfully formed spindles, 18% showed chromosome mass separation, a significant decrease compared to the Paclitaxel and solvent-treated RNAi control oocytes and similar to the results from BN2 treatment of Pp1-87B RNAi oocytes (Fig 4F and 4G). This rescue of chromosome mass separation demonstrates that PP1-87B antagonizes Aurora B in regulating chromosome organization. On the contrary, the sister centromeres remained separated in these oocytes ( Fig 4D and 4E, mean = 11.1), suggesting that stabilizing microtubule dynamics in Pp1-87B RNAi oocytes can override any effect of inhibiting Aurora B on sister centromere fusion. Based on these observations, we propose that PP1-87B regulates sister centromere separation by regulating microtubules dynamics. However, we cannot rule out the possibility that Aurora B is also required for centromere separation independently of the microtubules.

Kinetochore-microtubule interactions regulate chromosome mass organization and sister centromere fusion
The meiotic spindle consists of overlapping microtubules, only a portion of which make contact with the kinetochores. To understand which set of microtubules affect PP1-dependent centromere separation and chromosome mass disorganization, we used knockdowns of kinetochore proteins to specifically abrogate one class of microtubule contracts with the chromosomes. In Drosophila oocytes, SPC105R is required for lateral attachments and the localization of NDC80 whereas NDC80 is required for end-on attachments [20]. Thus, we co-depleted PP1-87B and SPC105R (no MT attachments) or NDC80 (lateral MT attachments only) and examined the chromosomes and centromeres. We found that loss of SPC105R, but not NDC80, suppressed the separated chromosome mass phenotype of Pp1-87B RNAi oocytes (Fig 5A and 5C), suggesting that the separated chromosome mass phenotype in Pp1-87B RNAi oocytes depends on lateral KT-MT interactions. The sister centromeres are already separated in Spc105R RNAi oocytes, and co-depletion of both Pp1-87B and Spc105R did not enhance the effects of either single knockdowns (Fig 5A and 5B). In contrast, the centromere separation phenotype was rescued in Ndc80, Pp1-87B double RNAi oocytes (mean = 9.0, Fig 5A and 5B) but not chromosome mass disorganization. We conclude that PP1-87B affects chromosome mass organization through regulating lateral KT-MT attachments and sister centromere fusion through end-on attachments.

PP1-87B antagonizes Polo and BubR1 in regulating sister centromere fusion
To identify proteins that function with PP1-87B in regulating end-on KT-MT attachments, we depleted proteins with meiotic functions that are involved in regulating microtubule attachments. Polo kinase localizes to centromeres in Drosophila metaphase I oocytes [40] (S3 Fig.), and in other organisms has been reported to stabilize KT-MT attachments [41][42][43][44]. Unlike Polo in mice [13], Drosophila polo RNAi oocytes do not show precocious sister centromere separation at metaphase I [45]. We depleted polo with RNAi in either Spc105R or Pp1-87B RNAi oocytes. Interestingly, we found that centromere separation in both mutant oocytes were rescued by polo RNAi (Fig 6A and 6B, mean = 6.6 and mean = 6.9). These results indicate that Polo negatively regulates both the separase-dependent (through SPC105R) and the microtubule attachment dependent pathways (through PP1-87B) for sister centromere fusion in Drosophila.
Two proteins, BubR1 and MPS1, function along with Polo to regulate KT-MT attachments in several organisms [41,42,46,47]. We predicted that depletion of either one could have a similar effect on the Pp1-87B oocyte phenotype as polo RNAi. Centromere separation in Pp1-87B RNAi oocytes was suppressed by simultaneous knockdown of BubR1 (Fig 6A and 6C; mean = 8.5) but not mps1 (Fig 6A and 6C; mean = 10.7). A caveat to this negative result is that, based on the non-disjunction rate, MPS1 is only partially depleted in these females (NDJ = 11%, n = 961, compared to a strong mps1 loss of function mutant, NDJ = 20.2%, n = 231 [48]). Regardless, these results suggest that PP1-87B promotes sister centromere fusion by antagonizing the activities of Polo and BubR1. In contrast, the frequency of oocytes with a separated chromosome mass phenotype remained similar to Pp1-87B RNAi oocytes when PP1-87B were co-depleted with BubR1 (Fig 6D), consistent with the results with NDC80. This result confirms that the separated chromosome mass phenotype in Pp1-87B RNAi oocytes depends on lateral KT-MT interactions. We propose that PP1-87B destabilizes end-on microtubule attachments by antagonizing Polo and BubR1 activities. In support of this conclusion, the increased spindle volume observed in of Pp1-87B RNAi oocytes was suppressed by co-depletion of polo or BubR1 ( Fig  6E). In summary, several experiments, including drug treatment (Paclitaxel+BN2), depletion of genes that affect KT-MT attachments, and measurements of spindle volume, support the conclusion that PP1-87B regulates KT-MT attachments, and these activities then affect sistercentromere separation and chromosome mass organization. The transverse element of the synaptonemal complex, C(3)G, is required for release of sister centromere fusion As described above, simultaneous loss of co-orientation and chiasmata can result in bi-orientation of univalent at meiosis I. We observed this phenomenon with simultaneous depletion of PP1-87B and mei-P22. The same experiment was done with c(3)G, which encodes a transverse element of the synaptonemal complex (SC) [49], because it is also required for crossing over [50]. Compared to mei-P22, however, we got surprisingly different results. First, c(3)G mutant females that were depleted of Pp1-87B failed to produce mature oocytes. We currently do not know why loss of c(3)G and prophase depletion of PP1-87B causes a failure in oocyte development, but it suggests C(3)G has a function in mid-oogenesis after its role in crossing over.
To examine the interaction between C(3)G and PP1-87B, c(3)G RNAi was used. To test the efficiency of the c(3)G RNAi, nanos-VP16-GAL4 was used to express the shRNA during early prophase, the frequency of X chromosome non-disjunction (NDJ) was similar to that observed in c(3) G null alleles (31%, n = 1647) [50]. In addition, C(3)G localization was absent in the germarium (S4 Fig.). These results suggest that this shRNA knockdown recapitulates the null mutant phenotype. For the double depletion we used mata4-VP16-GAL that induced shRNA expression later in oogenesis than nanos-VP16-GAL4. This was necessary because early expression of Pp1-87B shRNA results in a failure to produce oocytes. When using mata4-VP16-GAL to express shRNA, C(3)G was present in pachytene, crossing over was not affected (NDJ = 0%, n = 427), but C(3)G was missing from mid-late prophase (Fig 7A, S4 Fig.). These results indicate C(3)G is dynamic throughout prophase, and allows us to test if there is a late prophase-metaphase interaction between C(3)G and PP1-87B. Interestingly, RNAi of c(3)G rescued the sister centromere separation phenotype in Pp1-87B, but not Spc105R RNAi oocytes (Fig 7A and 7B). These results suggest that PP1-87B antagonizes centromeric C(3)G, after most of the SC has been disassembled, to maintain sister centromere fusion at metaphase I. As with other proteins that regulate end-on attachments, the Pp1-87B RNAi increased spindle volume phenotype was rescued to wild type levels by co-depletion of c(3)G (Fig 7C).
It is noteworthy that C(3)G is enriched at the centromere regions [51,52] in pachytene, although its function there is not known. In this location, and because C(3)G has a Polo-binding box, it is possible that C(3)G recruits Polo to the centromere region to regulate microtubule dynamics. However, when examining the localization of Polo in c(3)G RNAi oocytes, we did not observe any changes in protein localization compared to wild-type (S4 Fig.). Whether C(3)G plays a role in regulating microtubule dynamics through Polo or other independent function to regulate sister centromere fusion needs further investigation.

Discussion
The fusion of sister centromeres is important for co-orientation in meiosis I, ensuring that sister kinetochores attach to microtubules from the same pole. Release of this attachment must occur early in meiosis II. Based on our results, we propose that the regulation of sister centromere fusion that ensures its release in meiosis II occurs through at least two mechanisms (Fig 7D). First, Drosophila centromere fusion depends on loading cohesins at the centromeres that is protected by a kinetochore protein, SPC105R. Second, sister centromere fusion is released in a Separase-independent manner that depends on KT-MT interactions and is inhibited by PP1-87B.

Sister centromere fusion depends on kinetochore protein SPC105R to protect cohesion from Separase
Assembly of meiosis-specific cohesins at the centromeres probably establishes sister centromere fusion [2]. Indeed, the meiosis-specific cohesin complex SMC1/SMC3/SOLO/SUNN is enriched at Drosophila meiotic centromeres and could to have this function [14][15][16][17]. Guo et al found that separase is required for progression through both meiotic divisions in oocytes [53]. We found that depleting Separase in metaphase I Drosophila oocytes rescued the precocious centromere separation phenotype caused by loss of SPC105R. Although we found no role for WAPL in centromere fusion, we did not rule out other functions in meiosis, especially in anaphase I given that Guo et al. found that oocytes depleted for Separase were delayed in anaphase I but eventually progressed to meiosis II.
SPC105R may protect centromere cohesion by recruiting cohesin protection proteins such as MEI-S332/SGO that subsequently recruit PP2A. The fact that mei-S332 mutants do not display defects in meiosis I [32,33] could be due to redundancy with another Drosophila PP2A recruiter, Dalmatian [34,35]. The previous finding that Drosophila Spc105R mutants enhance defects in Separase function suggest SPC105R may have a cohesion protection function in other cell types [54]. However, Separase activation usually coincides with the entry into anaphase when the APC degrades an inhibitor of Separase, Securin [30]. One explanation is that Separase has a novel cohesin-independent function in regulating co-orientation through SPC105R, such as structural or regulatory function within the kinetochore or spindle [55], or that loss of SPC105R activates Separase. We favor, however, the explanation that Separase is active prior to anaphase I and cohesion is maintained only by PP2A activity in metaphase I arrested oocytes. This model can explain why knockout of SPC105R in male meiosis does not show a loss of centromere fusion [56]. In male meiosis where there is no cell cycle arrest, Separase may not be active until anaphase, which would make a protective role for SPC105R difficult to observe.

The transition of sister centromeres from co-orientation to bi-orientation depends on kinetochore-microtubule interactions
Aurora B inhibitor BN2 was applied to mature Pp1-87B RNAi oocytes, which were in prometaphase I or metaphase I and therefore, after the spindle had formed and the sister centromeres had separated. Because this treatment caused the sister centromeres to come back together, sister centromere separation in Pp1-87B RNAi oocytes appears to be reversible. In contrast, treatment of mature oocytes with BN2 did not reverse centromere separation in Spc105R RNAi. This reversible phenotype of Pp1-87B RNAi oocytes is consistent with a mechanism that involves the reorganization of centromere and kinetochore geometry, and the nonreversible phenotype of Spc105R RNAi with a mechanism that involves the degradation of cohesins. Furthermore, the results from destabilizing microtubule attachments with BN2 treatment suggested that centromere separation in PP1-87B-depleted oocytes depends on KT-MT interactions. In support of this conclusion, we found that PP1-87B affects several spindlebased parameters: it localizes to the meiotic spindle, its knockdown caused an increase in spindle volume, and centromere separation in PP1-87B-deplated oocytes depended on NDC80, Polo and BubR1. These results suggest that stable end-on attachments are required for release of sister centromere fusion. Similar conclusions have been made in Drosophila male meiosis. Sister centromere separation in meiosis II does not depend on Separase [57] but does depend on KT-MT interactions [56,58]. These findings are not limited to Drosophila. Classic micromanipulation experiments in grasshopper cells demonstrated that the switch in meiosis II to separated sister kinetochores requires attachment to the spindle [59]. Based on all these results, we propose that sister centromeres normally separate early in meiosis II by a process that is Separase-independent but microtubule-dependent (Fig 7D).
Interestingly, univalents in meiosis I can biorient in co-orientation-defective mutants that lack crossovers [13]. We observed a similar phenomenon in Pp1-87B; mei-P22 meiosis I oocytes. However, the frequency of univalent bi-orientation was low, raising the question of how meiosis II univalents preferentially achieve bi-orientation. The low frequency of univalent bi-orientation in meiosis I could be due to differences in how each division begins. Meiosis I begins with the centromeres clustered in a chromocenter and rapidly develops a robust central spindle, both of which may bias the sister centromeres to make attachments to the same pole, even in a PP1-87B knockdown oocyte (S5 Fig.).
The mechanism regulated by PP1-87B that regulates KT-MT interactions and maintains sister centromere fusion is not known and may be a function utilized in mitotic cells. For example, PP1 has a role in regulating microtubule dynamic in Xenopus extracts [60]. In HeLa cells depleted of SDS22, a regulatory subunit of PP1, sister kinetochore distances increase [61], similar to the defect we described here. In budding yeast, suppressing premature formation of stable kinetochore-microtubule attachments is necessary for co-orientation [62]. The mechanism may be related to the role of PP1 in negatively regulating condensin functions that affect chromosome structure [63,64]. A negative effect on condensin activity, which is known to shape mitotic chromosomes [65,66], could also explain the chromosome mass separation phenotype of Pp1-87B RNAi oocytes.
Our observations are strikingly similar to the phenomenon of cohesin fatigue, where sister chromatids separate in metaphase arrested mitotic cells. Identical to the effect of PP1-87B on centromere separation, cohesion fatigue occurs in a Separase-independent but microtubuledependent manner [67,68], however, the mechanism is unknown [69]. Oocytes with a prolonged arrest points, such as metaphase I in Drosophila, might prevent cohesion fatigue by concentrating meiotic cohesins at the centromeres and destabilizing KT attachments to reduce MT forces. In Drosophila oocytes, the microtubule catastrophe protein Sentin destabilizes endon KT-MT attachments after the spindle is well established [27]. In fact, active destabilization of kinetochore attachments may be a common feature of oocyte meiosis. Mammalian oocytes also have an extended period of dynamic KT-MT interactions [70], lasting 6-8 hours in mice and up to 16 hours in human [71,72]. All of these results are in line with our conclusion that oocytes require PP1-87B to prevent premature stable KT-MT attachments and avoiding cohesion fatigue.

On the role of C(3)G and Polo kinase in cohesion and co-orientation
Depletion of C(3)G suppresses the Pp1-87B centromere fusion defect. This result suggests that centromeric SC has a role in negatively regulating sister centromere co-orientation. While the bulk of SC disassembles in late prophase [73,74], centromeric SC proteins persist beyond pachytene in Drosophila and until at least metaphase I in budding yeast and mouse [51,[74][75][76][77]. It has also been shown that SC proteins interact with the NDC80 complex in two yeast two hybrid experiments [78,79]. These studies have concluded that centromeric SC is required for bi-orientation of homologs and monopolar attachment. Because both Polo Kinase and C (3)G negatively regulate co-orientation, we hypothesize that C(3)G could be required for Polo Kinase activity, but not localization, at the centromere. Thus, centromeric SC components might be an important mediator of co-orientation.
Co-orientation in yeast and mice depends on Polo kinase, which is recruited by Spo13, Moa1 or Meikin [80]. This is opposite of the known mitotic role of Polo in phosphorylating cohesin subunits and facilitating their removal from binding sister chromatids [31,81,82]. In yeast meiosis, however, the phosphorylation of cohesin subunits may depend on two different kinases, Casein kinase I and CDC7 [83][84][85]. Which kinase(s) are required in animals to phosphorylate meiotic cohesins for their removal remains unknown. We have shown that Polo is required for loss of centromeric cohesion, which to our knowledge is the first evidence of its kind in animal meiotic cells.
Unlike mice and yeast, depletion of Polo kinase from Drosophila metaphase I oocytes does not cause sister centromere separation [45]. One reason for this difference in Polo function could be that it is required at multiple stages of meiosis and its phenotype may depend on when it is absent. Loss of Polo or BubR1 during early Drosophila prophase (pachytene) oocytes leads to loss of SC and cohesion defects [86,87]. Our experiments depleted Polo after cohesion was established. Alternatively, the function of Polo in co-orientation may not be conserved. Importantly, two features of centromere fusion and co-orientation that are conserved are maintenance depending on SPC105R and separation depending on stabilization of KT-MT attachments. Like SPC105R in Drosophila, budding yeast KNL1 is required for meiotic sister centromere fusion and co-orientation and is a target of Polo [18]. The differences between Drosophila and mouse or yeast can be explained if SPC105R does not require Polo in order to protect cohesion at the centromeres for co-orientation.
While all previous studies of co-orientation have focused on the establishment of centromere fusion, our results identified several key regulators and provide insights into how sister centromere fusion is maintained in meiosis I and released for meiosis II. In contrast to release of cohesion in most regions of the chromosomes, we propose a Separase-independent mechanism that requires stable kinetochore-microtubule attachments promotes centromere separation early in meiosis II. While it is well known that regulating microtubule attachments is important for bi-orientation, our results are an example of another reason why KT-MT attachments must be properly regulated, to safely navigate the transitions through the two divisions of meiosis.

Drosophila genetics
Drosophila were crossed and maintained on standard media at 25˚C.

RNAi in oocytes: expression and quantification
Most Drosophila lines expressing a short hairpin RNA were designed and made by the Transgenic RNAi Project, Harvard (TRiP) ( Table 1). To deplete target mRNA, a cross was performed to generate females carrying both the UAS:shRNA and a GAL4-VP16 transgene. The shRNA can be induced ubiquitous expression by crossing to tubP-GAL4-LL7 and testing lethality [89], or mata4-GAL-VP16 and osk-GAL4-VP16 for oocyte-specific expression [90]. In this paper, mata4-GAL-VP16 was primarily used for inducing expression of the UAS:shRNA after early pachytene but throughout most stages of oocyte development in the Drosophila ovary. This allows for 3-5 days of continuous expression to knockdown the mRNA levels. In some cases, we used the oskar -GAL4-VP16 transgene [91,92], which causes a similar knockdown and phenotype in PP1-87B as mata4-GAL-VP16. Double RNAi crosses were set up based on the available RNAi lines ( Table 2). For measuring the mRNA knockdown level, total RNA was extracted from late-stage oocytes using TRIzol Reagent (Life Technologies) and reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qPCR was performed on a StepOnePlus (Life Technologies) real-time PCR system using TaqMan Gene Expression Assays (Life Technologies), Dm02152292_g1 for Pp1-87B and Dm02134593_g1 for the control RpII140. Oocyte-specific shRNA expression of HMS00409 using mata4-GAL-VP16 resulted in sterility and knockdown of the oocyte mRNA to 35% as measured by RT-qPCR; the same phenotype has been seen when using osk-GAL4-VP16, where the mRNA knockdown is also to 35%. For SPC105R, expressing shRNA GL00392 using osk-GAL-VP16 knocked down the mRNA to 10%.

Generation of Wapl shRNA lines in Drosophila
To generate a wapl shRNA line, we followed the protocol in Harvard TRiP center (http://fgr. hms.harvard.edu/trip-plasmid-vector-sets) and targeted wapl sequence (5'-gaggaggaggatcaacagcaa -3') for mRNA knockdown. This 21-nucleotide sequence was cloned into pVALIUM22 and the whole construct was injected into Drosophila embryos (y sc v; attP40). The mRNA is knocked down to 4% when using mata4-GAL-VP16 to express the shRNA in oocytes.

Antibodies and immunofluorescent microscopy
Mature (stage 12-14) oocytes were collected from 100 to 200, 3-4-day old yeast-fed non-virgin females. The procedure is described as in [93]. Oocytes were stained for DNA with Hoechst

Image analysis
All the CENP-C foci, CID foci, chromosome mass volume, spindle volume and MEI-S332 volume were measured using Imaris image analysis software (Bitplane). For determining centromere foci, an automated spots detection function in Imaris was used. A spot whose XY diameter is 0.20 μm, Z diameter is 1.00 μm and is in touch with DNA will be counted as a centromere. For the volume measurement, images were resampled first to become isovoxel data. Then the surface detection function was used for defining different objects and measuring their volume. Spindle volume is measured and normalized by the chromosome mass volume to compensate for effects of chromatin volume on microtubule recruitment, although chromosome mass volume in each genotype did not differ significantly.

Quantification and statistical analysis
Statistical tests were performed using GraphPad Prism software. All the numbers of the centromere foci or spindle/chromosome mass volume or MEI-S332 volume were pooled together and ran one-way ANOVA followed by post hoc pairwise Tukey's multiple comparison test. Details of statistical evaluations and the numbers of samples are provided in the figure legends. To observe whether the microtubule attachments in Pp1-87B RNAi oocytes are merotelic or syntelic in metaphase I, we used cold treatment to remove the unstable attachments. All females were cold treated for 2 hours before fixation. Presumably because depletion of PP1-87B stabilizes microtubule attachments, the Pp1-87B RNAi oocytes show a partial resistance to cold-treatment compared to wild-type. The images were taken and processed through deconvolution. All images are maximum projections and scale bars are 5 μm.