Cohesin and microtubule dependent mechanisms regulate sister centromere fusion during meiosis I

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. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) are required for this process. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion during meiosis I in Drosophila oocytes. Double depletion experiments demonstrated that the precocious separation of centromeres in Spc105R RNAi oocytes is Separase-dependent, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, precocious sister centromere separation in Pp1-87B RNAi oocytes does not depend on Separase or Wapl. Further analysis with microtubule destabilizing drugs showed that PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. Additional double depletion experiments demonstrated that PP1-87B has this function by antagonizing Polo kinase and BubR1, two proteins known to promote kinetochore-microtubule (KT-MT) attachments. These results suggest that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, we found that loss of C(3)G, the transverse element of the synaptonemal complex (SC), suppresses centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions. 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. Author Summary Meiosis involves two cell divisions. In the first division, pairs of homologous chromosomes segregate, in the second division, the sister chromatids segregate. These patterns of division are mediated by regulating microtubule attachments to the kinetochores and stepwise release of cohesion between the sister chromatids. During meiosis I, cohesion fusing sister centromeres must be intact so they attach to microtubules from the same pole. At the same time, arm cohesion must be released for anaphase I. Upon entry into meiosis II, the sister centromeres must separate to allow attachment to opposite poles, while cohesion surrounding the centromeres must remain intact until anaphase II. How these different populations of cohesion are regulated is not understood. We identified two genes required for maintaining sister centromere cohesion, and surprisingly found they define two distinct mechanisms. The first is a kinetochore protein that maintains sister centromere fusion by recruiting proteins that protect cohesion. The second is a phosphatase that antagonizes proteins that stabilize microtubule attachments. We propose that entry into meiosis II coincides with stabilization of microtubule attachments, resulting in the separation of sister centromeres without disrupting cohesion in other regions, facilitating attachment of sister chromatids to opposite poles.


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 coorientation 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 corecentromere 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 Sphase and are enriched at the core centromeres [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 coorientation. 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 karyosome organization sister centromere fusion in meiosis I
Drosophila has three homologs of the mammalian Phosphatase1 (PP1α/γ) genes, Pp1-87B, Pp1-96A and Pp1-13C. 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 spherical structure, the karyosome, at the center of the spindle ( Figure 1A). In 62% of Pp1-87B RNAi oocytes, the karyosome was separated into multiple groups of chromosomes ( Figure 1A, B). 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 ( Figure 1A, C). 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 separate karyosome 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 homologs. In wild type, two FISH foci are typically observed per homologous chromosome pair, oriented towards opposite poles but within a single karyosome ( Figure 1D). If there were loss of arm cohesion, the two homologs could separate and be observed as two FISH foci in separate chromosome masses.
We observed that while 62% of Pp1-87B RNAi oocytes (n = 50) had separated karyosomes, 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 karyosome phenotype in Pp1-87B RNAi oocytes is due to intact bivalents failing to organize correctly at the center of the spindle.
The same FISH probes could be used 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 (mean = 2.7, Figure 1E). In contrast, the average number of dodeca foci in Pp1-87B RNAi oocytes was not significantly higher than wild type ( Figure 1E; mean = 3.0), suggesting that pericentromeric cohesion is intact in Pp1-87B RNAi oocytes. From these FISH assays we conclude that 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.
The defects in Pp1-87B RNAi oocytes do result in errors in bi-orientation. In the FISH experiments we observed 5.3% of the homologs in Pp1-87B RNAi oocytes were mono-oriented (n=130 v.s. nwt=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 biorient.
The increased number of centromere foci in Pp1-87B RNAi oocytes suggests a defect in sister centromere fusion at meiosis I and hence a lack of co-orientation. When sister centromeres 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, 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 bioriented. Indeed, we observed that 20% of mei-P22, Pp1-87B RNAi oocytes had sister chromatids bi-oriented (n = 15, Figure 1F). These results suggest that PP1-87B is required for sister centromere fusion and this facilitate co-orientation in metaphase I of oocytes.

Co-orientation in Drosophila oocytes requires both cohesin-dependent and cohesinindependent 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 [26]. To investigate if cohesin is involved in sister centromere fusion in, 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 Apart-like (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 (Figure 2A, B), suggesting that Wapl does not regulate sister centromere fusion. While centromere protein localization in wapl, Spc105R RNAi oocytes was normal, centromere localization in wapl, Pp1-87B RNAi oocytes became thread-like instead of punctate (Figure 2A), leading to additional centromere foci when quantified ( Figure 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 [27,28].
The separated centromere phenotype was rescued in sse, Spc105R RNAi oocytes ( Figure   2A, C; 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 during meiotic metaphase I [29]. If Separase is active, these results could be explained if SPC105 recruits proteins that protect cohesins from Separase. To test the hypothesis that SPC105R protects cohesins from Separase, we examined the localization of the cohesion protection protein MEI-S332. MEI-S332 localizes to centromere and peri-centromeric regions in wild-type oocytes, as shown by colocalization and substantial non-overlap distribution with the core centromere ( Figure S 1). MEI-S332 localization was almost abolished in Spc105R RNAi oocytes ( Figure   2D). This result suggests 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 (Figure 2A, C; mean= 13.4). Consistent with cohesin-independence of these phenotypes, the intensity of MEI-S332 in Pp1-87B RNAi oocytes was not reduced, and in fact, was increased relative to wild-type ( Figure   2D). Aurora B is required for MEI-S332 localization [30], and our results suggest MEI-S332 localization is constrained by antagonism between PP1-87B and Aurora B. 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 ( Figure 3A). In addition, PP1-87B was found to localize to the oocyte meiotic spindle (Figure S 2). 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 [31] and can be antagonized by PP1 in other systems [32]. Furthermore, they have opposite phenotypes: both karyosome and sister centromeres preciously separate in Pp1-87B RNAi oocytes but remain together in Aurora B-depleted oocytes [31]. Therefore, we tested whether Aurora B is required for both the karyosome and centromere separation phenotypes of Pp1-87B RNAi oocytes.
Treatment of oocytes with the Aurora B inhibitor, Binucleine 2 (BN2) [33], caused loss of the spindle (65%, n=29, Figure 3B and C), consistent with previous findings that Aurora B is required for spindle assembly [31]. However, Pp1-87B RNAi oocytes showed resistance to BN2 treatment; only 9% had lost the spindle and 50% of oocytes had residual MT around the karyosome (n= 22, Figure 3B and C). 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 ( Figure 3D and E, mean = 7.4) similar to wild-type controls ( Figure 3D and E, mean = 7.7).
Similarly, the increased frequency of karyosome separation in Pp1-87B RNAi oocytes was rescued by BN2 treatment ( Figure 3F and G). In contrast, centromere separation was not rescued by BN2 treatment of Spc105R RNAi oocytes ( Figure 3B and C, 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. These oocytes successfully formed spindles; however, 18% showed karyosome separation, a significant decrease compared to the Paclitaxel and solventtreated RNAi control oocytes and similar to the results from BN2 treatment of Pp1-87B RNAi oocytes ( Figure 3F and G). This rescue of karyosome separation demonstrates that PP1-87B antagonizes Aurora B in regulating karyosome organization. However, the sister centromeres remained separated in these oocytes ( Figure 3D and E, 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 karyosome organization and sister centromere fusion
The meiotic spindle consists of overlapping microtubules, only a portion of which make contact with kinetochore. To understand which set of microtubules affect PP1-dependent centromere separation and karyosome 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 karyosome phenotype of Pp1-87B RNAi oocytes ( Figure 4A and C), suggesting that the separated karyosome phenotype in Pp1-87B RNAi oocytes depends on lateral KT-MT interactions. The sister centromeres are already separated in Spc105R RNAi oocytes, and codepletion of both Pp1-87B and Spc105R did not enhance the effects of either single knockdowns ( Figure 4A, B). In contrast, the centromere separation phenotype was rescued in Ndc80, Pp1-87B double RNAi oocytes (mean = 9.0, Figure 4A, B) but not karyosome disorganization. We conclude that PP1-87B affects karyosome 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 [34] (Figure S 3), and in other organisms has been reported to stabilize KT-MT attachments [35][36][37][38]. Unlike Polo in mice [13], Drosophila polo RNAi oocytes do not show precocious sister centromere separation at metaphase I [39]. 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 (Figure 5A and B, mean = 6.6 and mean = 6.9).
These results indicate that Polo negatively regulates both the cohesion-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 [35,36,40,41]. 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 We propose that PP1-87B inhibits 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 ( Figure 5E). 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 sister-centromere separation and karyosome 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 biorientation of univalent at meiosis I. We observed this phenomenon with codepletion 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) [43], because it is also required for crossing over [44]. Compared to mei-P22, however, we got surprisingly different results.  Figure 6A and B). 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 ( Figure 6C).
It is noteworthy that C(3)G is enriched at the centromere regions [45,46]

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 ( Figure 6D). 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.
Depleting Separase in metaphase I Drosophila oocytes rescued the precocious centromere separation phenotype caused by loss of SPC105R. Thus, SPC105R protects centromere cohesion, probably by recruiting cohesin protection proteins such as MEI-S332/SGO that subsequently recruit PP2A. A previous finding that Drosophila Spc105R mutants enhance defects in Separase function suggest SPC105R may have this function in other cell types [47]. However, Separase activation usually coincides with the entry into anaphase when the APC degrades an inhibitor of Separase, Securin [29]. One possible explanation is 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 [48]. 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
Several of our experiments demonstrated that centromere separation in Pp1-87B RNAi oocytes was reversible. In addition, treatment of prometaphase I oocytes with the Aurora B inhibitor BN2 showed that centromere separation is reversible in Pp1-87B RNAi but not Spc105R RNAi. This reversible phenotype is consistent with a mechanism that involves the reorganization of centromere and kinetochore geometry in Pp1-87B RNAi oocytes rather than 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 spindle-based 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 [49] but does depend on KT-MT interactions [48,50]. 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 [51]. 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 ( Figure 6D).
The mechanism regulated by PP1-87B that regulates KT-MT interactions and maintains sister centromere fusion may be a function utilized in mitotic cells. For example, PP1 has a role in regulating microtubule dynamic in Xenopus extracts [52]. In HeLa cells depleted of SDS22, a regulatory subunit of PP1, sister kinetochore distances increase [53], similar to the defect we described here. In budding yeast, suppressing premature formation of stable kinetochoremicrotubule attachments is necessary for co-orientation [54]. Finally, 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 microtubule-dependent manner [55,56], however, the mechanism is unknown [57].
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 end-on KT-MT attachments after the spindle is well established [26]. 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 [58], lasting 6-8 hours in mice and up to 16 hours in human [59,60]. 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 [61,62], centromeric SC proteins persist beyond pachytene in Drosophila and until at least metaphase I in budding yeast and mouse [45,[62][63][64][65].
It has also been shown that SC proteins interact with NDC80 complex in two yeast two hybrid experiments [66,67]. These studies have concluded that centromeric SC is required for biorientation 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 [68]. This is opposite of the known mitotic role of Polo in phosphorylating cohesin subunits and facilitating their removal from binding sister chromatids [69][70][71]. In yeast meiosis, however, the phosphorylation of cohesin subunits may depend on two different kinases, Casein kinase I and CDC7 [72][73][74]. 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 [39]. 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 [75,76]. Our experiments deplete Polo after cohesion is 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 and 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 to promotes centromere separation early in meiosis II. While it is well known that regulating microtubule attachments is important for bi-orientation, our result 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 wss 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 [78], or mata4-GAL-VP16 and osk-GAL4-VP16 for oocyte-specific expression [79]. 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 [80,81], 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 [82]. Oocytes were stained for DNA with Hoechst 33342 (10 µg/ml) and for MTs with mouse anti-α tubulin monoclonal antibody DM1A (1:50), directly conjugated to FITC (Sigma, St. Louis). Additional primary antibodies used were rat anti-Subito antibody [34], rat anti-INCENP [83], guinea pig anti-MEI-S332 [84], rabbit anti-CENP-C [85], rabbit anti-Deterin [86], rabbit anti-Spc105R [87], mouse anti-Polo [88] and rabbit anti-CID (Active Motif). These primary antibodies were combined with either a Cy3, Alex 594 or Cy5 secondary antibody pre-absorbed against a range of mammalian serum proteins (Jackson Immunoresearch, West Grove, PA). FISH probes corresponding to the X359 repeat labeled with Alexa 594, AACAC repeat labeled with Cy3 and the dodeca repeat labeled with Cy5 were obtained from IDT. Oocytes were mounted in SlowFade Gold (Invitrogen). Images were collected on a Leica TCS SP8 confocal microscope with a 63x, NA 1.4 lens. Images are shown as maximum projections of complete image stacks followed by merging of individual channels and cropping in Adobe Photoshop (PS6). CENP-C foci, CID foci, karyosome volume and spindle volume were measured using Imaris image analysis software (Bitplane) and graphs were plotted and statistics calculated using Graphpad Prism software.

Quantification and statistical analysis
Statistical tests were performed using GraphPad Prism software. All the numbers of the centromere foci or spindle/karyosome volume were pooled together and ran one-way ANOVA