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

  • Lin-Ing Wang,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing

    Affiliation Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey, United States of America

  • Arunika Das,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing

    Current address: University of Pennsylvania, Philadelphia, United States of America

    Affiliation Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey, United States of America

  • Kim S. McKim

    Roles Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review & editing

    Affiliation Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey, United States of America


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.

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.


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 [35]. 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 [1417]. 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 cohesin-independent 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.

Fig 1. Pp1-87B RNAi oocytes have defects in chromosome mass organization and sister centromere fusion.

(A) Pp1-87B RNAi oocytes show separated chromosome mass and sister centromere (red) separation in metaphase I with tubulin in green and DNA in blue. DNA and centromeres are shown in separate channels. In wild-type, the fourth chromosomes sometimes appear as a dot separated from the chromosome mass. Scale bars indicate 5 μm. (B) Quantification of the separated chromosome mass phenotype in wild-type (n = 20) and Pp1-87B RNAi oocytes (n = 50). **** = p < 0.0001. Error bars indicate 95% confidence interval. (C) Quantification of centromere foci. Error bar shows standard deviation. Number of oocytes: wild type n = 16 and Pp1-87B RNAi n = 27. **** = p < 0.0001, (D) Chromosome mass separation defect in Pp1-87B RNAi oocytes. DNA channel is shown in the inset. FISH probes for the X (359 bp repeat, purple), 2nd (AACAC, red) and 3rd chromosome (dodeca, white) were used to detect pericentromeric heterochromatin. The chromosome mass is outlined in white. Two examples are shown of Pp1-87B RNAi oocytes with homologs bi-oriented and not bi-oriented. Scale bars are 5 μm. (E) Quantification of dodeca foci to detect precocious separation of pericentromeric heterochromatin. Number of oocytes: wild-type (n = 27), ord (n = 15), mei-S332 (n = 14), Pp1-87B RNAi (n = 50) and Spc105R RNAi (n = 21). **** = p < 0.0001. (F) Recombination defective mutant mei-P22 displayed homologous chromosome separation indicting precocious anaphase I in oocytes normally arrested in metaphase I. Knocking down Pp1-87B in a mei-P22 mutant background resulted in sister chromatid bi-orientation in meiosis I (arrow). The bioriented univalent can be identified because it has not segregated to a pole like the other univalents. In addition, it is the only chromosome where the centromeres are oriented towards opposite poles. And given that there are no chiasmata, the only linkage between these two centromeres can be pericentromeric cohesion of sister chromatids.

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 (mean = 2.7, Fig 1E). In contrast, the average number of dodeca foci in Pp1-87B RNAi oocytes was not significantly higher than wild type (Fig 1E; mean = 3.0), suggesting that pericentromeric cohesion is intact in Pp1-87B RNAi oocytes. Secondly, we used these FISH probes to test if there were loss of arm cohesion, defined as when the homologs separate and are observed as 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. 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 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 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 (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].

Fig 2. Sister centromere fusion defect rescued by loss of Separase in Spc105R RNAi but not Pp1-87B RNAi oocytes.

(A) Confocal images showing the centromeres (red) in wild-type, sse RNAi, and wapl RNAi in combination with Pp1-87B RNAi or Spc105R RNAi. Centromeres are shown in separate channel. Scale bars are 5 μm. (B, C) Dot plots summarize the quantification of centromere foci number in (A). Error bars indicate standard deviation, ** = p <0.01, *** = p <0.001 and **** = p <0.0001. Number of oocytes are wapl RNAi (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).

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 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.

Fig 3. MEI-S332 localizes to centromeres and heterochromatin.

A) MEI-S332 has enhanced recruitment to the pericentromeric regions in Pp1-87B RNAi oocytes and is decreased in Spc105R RNAi oocytes. Two images of Spc105R RNAi oocytes show MEI-S332 localization either abolished or greatly reduced. Confocal images are shown with centromeres (white), MEI-S332 (red), tubulin (green) and DNA (blue). Scale bar indicates as 5 μm. (B) Quantification of MEI-S332 volume. The number of oocytes for measuring are wild type (14), Pp1-87B RNAi oocytes (16) and Spc105R RNAi oocytes (16). Error bars indicate standard deviation, ** = p <0.01 and **** = p <0.0001.

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.

Fig 4. PP1-87B regulation of sister centromere fusion depends on microtubules.

(A) Graph showing the spindle volume relative to the chromosome mass volume. The chromosome mass volumes remain constant while Pp1-87B RNAi oocytes (n = 31) had increased spindle volume compared to wild type (n = 22) oocytes. **** = p <0.0001 (B) Wild-type, Pp1-87B RNAi and Spc105R RNAi oocytes treated with 50μM BN2 or the solvent for one hour. All images are shown with DNA (blue), tubulin (green) and centromeres (white), and the scale bars are 5 μm. (C) Quantification of spindle phenotype in wild-type (n = 28) and Pp1-87B RNAi (n = 22) oocytes after one hour of BN2 treatment. (D) Wild type and Pp1-87B RNAi oocytes treated with Paclitaxel for 10 minutes followed by either BN2 or DMSO for one hour. Centromeres are marked in white. Scale bars are 5 μm. (E) Quantification of centromere foci in indicated genotypes of oocytes (n = 15, 6, 29, 14, 12, 12, 17, 12, 15 and 13 in the order of the graph). Error bars show standard deviation and **** = p<0.0001. (F) Chromosome mass organization in Pp1-87B RNAi oocytes treated for 10 minutes in Paclitaxel followed by BN2 or DMSO for 30 minutes. INCENP localization is shown in red, DNA in blue and tubulin in green. The single channel of DNA is also shown. Scale bar = 5 μm. (G) Quantification from the same experiment in (F). Error bars indicate 95% confidence intervals and *** = p<0.001. The numbers of oocytes were 143,151, 21, 27, 111 in order of the graph.

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.

Fig 5. PP1-87B regulates chromosome alignment through lateral attachment and co-orientation via end-on attachment.

(A) Confocal images of Pp1-87B RNAi oocytes when expressing Spc105R RNAi or Ndc80 RNAi. Centromeres are in white, DNA is in blue and tubulin is in green. Single channel image is selected to show DNA in the merge images. Error bars = 5μm. (B) Dot plot shows the number of centromere foci in each genotype. Oocytes numbers are 27, 26, 20 and 19 in order of the graph. Error bars show standard deviation. ** = p<0.01. (C) Quantification of oocytes with a separated chromosome mass. Error bars indicate 95% confidence interval. Numbers of oocytes are 29, 20, and 19 in order of the graph. **** = p<0.0001.

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 [4144]. 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.

Fig 6. Polo and BubR1 antagonize PP1-87B effects on KT-MT interactions.

(A) Confocal images showing polo, BubR1 or mps1 RNAi expressed along with Spc105R RNAi or Pp1-87B RNAi in oocytes. DNA is in blue, tubulin is in green and centromeres are in red. Scale bars = 5μm. (B) Dot plot showing the number of centromeres foci in (A). Oocytes numbers are 26, 24, 27, and 20 in order of the graph. Error bars show standard deviation and **** = p<0.0001. (C) Dot plot showing the number of centromere foci in (A). Oocytes numbers are 26, 16, 21, 22, 12, 27, 22 and 19 in order of the graph. Error bars show standard deviation and *** = p<0.001. (D) Graph showing the percentage of a separated chromosome mass in oocytes depleted for a variety of kinases in the presence or absence of Pp1-87B RNAi. Error bars indicate standard deviation. Numbers of oocytes of each genotype are 20, 31, 21, 19, 22 in order of the graph. (E) Dot plot showing the spindle volume relative to the chromosome mass volume. Number of oocytes are: 22, 31, 20, 21, 19, 22, 12, 9 in order of the graph. Error bars show standard deviation ** = p<0.01 and **** = p<0.0001.

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 sister-centromere 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).

Fig 7. PP1-87B antagonizes C(3)G to regulate sister centromere fusion.

(A) Confocal images of oocytes expressing c(3)G RNAi in combination with Pp1-87B or Spc105R RNAi. The centromeres are shown in red in the merged images. Scale bar = 5 μm. (B) Dot plot showing the number of centromere foci in (A). Number of oocytes of each genotype are 14, 27, 18, 26 and 23 in order of the graph. Error bars indicates standard deviation. ** = p<0.01. (C) Graph showing the ratio of the spindle volume to the chromosome mass volume. Number of oocytes are: 22, 31, 20 and 17. Error bars show standard deviation, **** = p<0.0001. (D) Model for regulation of co-orientation in Drosophila oocytes.

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.


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 [1417]. 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 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 [57] but does depend on KT-MT interactions [56, 58]. These findings are not limited to Drosophila. Classic micro-manipulation 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 microtubule-dependent 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 end-on 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, 7477]. 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 [8385]. 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. Fly stocks were obtained from the Bloomington Stock Center or the Transgenic RNAi Project at Harvard Medical School [TRiP, Boston, MA, USA,, 23]. Information on genetic loci can be obtained from FlyBase [, 88].

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).

Table 1. Genotypes of Gal4 and shRNA lines used for RNAi experiments.

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 ( 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 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 [40], rat anti-INCENP [94], guinea pig anti-MEI-S332 [95], rabbit anti-CENP-C [96], rabbit anti-Deterin [97], rabbit anti-SPC105R [98], mouse anti-Polo [99] 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).

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.

Binuclein 2 treatment assay

To inhibit Aurora B, oocytes were incubated with either 0.1% DMSO or 50 μM BN2 in 0.1% DMSO for 60 minutes prior to fixation in Robb’s media. To stabilize MTs, oocytes were incubated with either 0.1% DMSO or 10 μM Paclitaxel (Sigma) in 0.1% DMSO for 10 minutes, followed by 50 μM BN2 plus 10 μM Paclitaxel in 0.1% DMSO for 60 minutes.

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.

Supporting information

S1 Fig. MEI-S332 localization does not co-localize with centromere.

Representitive picture of wild type oocytes staining MEI-S332 (red) and CID (white) is shown and measured the intensity of flourensent. MEI-S332 localizes to both the pericentromeric and centromeric regions. Scale bar is 5 μm.


S2 Fig. Localization of PP1-87B to meiotic spindle.

An epitope-tagged version of PP1-87B was expressed from a UASP transgene using mata4-GAL-VP16. HA-PP1-87B is in red, tubulin in green and DNA in blue and scale bars are 5 μm.


S3 Fig. Polo localization does not change in c(3)G RNAi oocytes but decreases in Spc105R RNAi oocytes.

Wild-type, c(3)G RNAi, and Spc105R RNAi oocytes with DNA in blue, tubulin in green, Polo in red and CID in white. Single channels are shown in white. All images are maximum projections and scale bars are 5 μm.


S4 Fig. C(3)G is knockdown by shRNA expressed in the germline.

(A) C(3)G (red) forms thread-like structure in the germarium (early prophase), and retains them in oocytes of stages 2–5 of the vitellarium (late prophase). (B) When nanos-VP16-GAL4 expressed c(3)G shRNA in early prophase, C(3)G expression was abolished. (C) When mata4-GAL-VP16 expressed c(3)G shRNA in late prophase, C(3)G localization was present in germarium early pachytene, but absent in the stages 2–5 of the vitellarium. Scale bars are 10 μm.


S5 Fig. Kinetochore-microtubule attachments in Pp1-87B RNAi oocytes.

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.



We thank Li Nguyen for technical assistance; Christian Lehner, Terry Orr-Weaver and Claudio Sunkel for providing antibodies and Karen Schindler, Ruth Steward and Sarah Radford for helpful comments on the manuscript and Consuelo Ibar for assistance with deconvolution software. We thank the TRiP at Harvard Medical School for providing transgenic RNAi fly stocks used in this study. Fly stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were also used in this study.


  1. 1. Watanabe Y. Geometry and force behind kinetochore orientation: lessons from meiosis. Nat Rev Mol Cell Biol. 2012;13(6):370–82. pmid:22588367.
  2. 2. Nasmyth K. A meiotic mystery: How sister kinetochores avoid being pulled in opposite directions during the first division. Bioessays. 2015;37(6):657–65. pmid:25874377.
  3. 3. Katis VL, Matos J, Mori S, Shirahige K, Zachariae W, Nasmyth K. Spo13 facilitates monopolin recruitment to kinetochores and regulates maintenance of centromeric cohesion during yeast meiosis. Curr Biol. 2004;14(24):2183–96. pmid:15620645.
  4. 4. Clyne RK, Katis VL, Jessop L, Benjamin KR, Herskowitz I, Lichten M, et al. Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I. Nat Cell Biol. 2003;5(5):480–5. pmid:12717442.
  5. 5. Lee BH, Amon A. Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation. Science. 2003;300(5618):482–6. pmid:12663816.
  6. 6. Corbett KD, Harrison SC. Molecular architecture of the yeast monopolin complex. Cell reports. 2012;1(6):583–9. pmid:22813733; PubMed Central PMCID: PMCPMC3494995.
  7. 7. Corbett KD, Yip CK, Ee LS, Walz T, Amon A, Harrison SC. The monopolin complex crosslinks kinetochore components to regulate chromosome-microtubule attachments. Cell. 2010;142(4):556–67. pmid:20723757; PubMed Central PMCID: PMCPMC2955198.
  8. 8. Li X, Dawe RK. Fused sister kinetochores initiate the reductional division in meiosis I. Nat Cell Biol. 2009;11(9):1103–8. pmid:19684578.
  9. 9. Yokobayashi S, Watanabe Y. The kinetochore protein Moa1 enables cohesion-mediated monopolar attachment at meiosis I. Cell. 2005;123(5):803–17. pmid:16325576.
  10. 10. Chelysheva L, Diallo S, Vezon D, Gendrot G, Vrielynck N, Belcram K, et al. AtREC8 and AtSCC3 are essential to the monopolar orientation of the kinetochores during meiosis. J Cell Sci. 2005;118(Pt 20):4621–32. pmid:16176934.
  11. 11. d'Erfurth I, Jolivet S, Froger N, Catrice O, Novatchkova M, Mercier R. Turning meiosis into mitosis. PLoS Biol. 2009;7(6):e1000124. pmid:19513101; PubMed Central PMCID: PMCPMC2685454 was deposited by INRA on 7 January 2009.
  12. 12. Sakuno T, Tada K, Watanabe Y. Kinetochore geometry defined by cohesion within the centromere. Nature. 2009;458(7240):852–8. pmid:19370027.
  13. 13. Kim J, Ishiguro K, Nambu A, Akiyoshi B, Yokobayashi S, Kagami A, et al. Meikin is a conserved regulator of meiosis-I-specific kinetochore function. Nature. 2015;517(7535):466–71. pmid:25533956.
  14. 14. Khetani RS, Bickel SE. Regulation of meiotic cohesion and chromosome core morphogenesis during pachytene in Drosophila oocytes. J Cell Sci. 2007;120(Pt 17):3123–37. pmid:17698920.
  15. 15. Gyuricza MR, Manheimer KB, Apte V, Krishnan B, Joyce EF, McKee BD, et al. Dynamic and Stable Cohesins Regulate Synaptonemal Complex Assembly and Chromosome Segregation. Curr Biol. 2016;26(13):1688–98. pmid:27291057; PubMed Central PMCID: PMCPMC4942336.
  16. 16. Yan R, McKee BD. The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis, Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis. PLoS Genet. 2013;9(7):e1003637. pmid:23874232.
  17. 17. Krishnan B, Thomas SE, Yan R, Yamada H, Zhulin IB, McKee BD. Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster. Genetics. 2014. pmid:25194162.
  18. 18. Miyazaki S, Kim J, Yamagishi Y, Ishiguro T, Okada Y, Tanno Y, et al. Meikin-associated polo-like kinase specifies Bub1 distribution in meiosis I. Genes Cells. 2017;22(6):552–67. pmid:28497540.
  19. 19. Bernard P, Maure JF, Javerzat JP. Fission yeast Bub1 is essential in setting up the meiotic pattern of chromosome segregation. Nat Cell Biol. 2001;3(5):522–6. pmid:11331883.
  20. 20. Radford SJ, Hoang TL, Gluszek AA, Ohkura H, McKim KS. Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes. PLoS Genet. 2015;11(10):e1005605. pmid:26473960; PubMed Central PMCID: PMCPMC4608789.
  21. 21. Kirchner J, Gross S, Bennett D, Alphey L. Essential, overlapping and redundant roles of the Drosophila protein phosphatase 1 alpha and 1 beta genes. Genetics. 2007;176(1):273–81. pmid:17513890; PubMed Central PMCID: PMCPMC1893066.
  22. 22. Dombrádi V, Axton JM, Brewis ND, da Cruz e Silva EF, Alphey L, Cohen PT. Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1. Eur J Biochem. 1990;194(3):739–45. pmid:2176604.
  23. 23. Ni JQ, Zhou R, Czech B, Liu LP, Holderbaum L, Yang-Zhou D, et al. A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nature methods. 2011;8(5):405–7. pmid:21460824.
  24. 24. Radford SJ, Hoang TL, Głuszek AA, Ohkura H, McKim KS. Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes. PLoS Genet. 2015;11(10):e1005605. pmid:26473960.
  25. 25. Shonn MA, McCarroll R, Murray AW. Spo13 protects meiotic cohesin at centromeres in meiosis I. Genes Dev. 2002;16(13):1659–71. pmid:12101124; PubMed Central PMCID: PMCPMC186364.
  26. 26. Liu H, Jang JK, Kato N, McKim KS. mei-P22 encodes a chromosome-associated protein required for the initiation of meiotic recombination in Drosophila melanogaster. Genetics. 2002;162:245–58. pmid:12242237
  27. 27. Głuszek AA, Cullen CF, Li W, Battaglia RA, Radford SJ, Costa MF, et al. The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes. J Cell Biol. 2015;211(6):1113–20. pmid:26668329; PubMed Central PMCID: PMCPMC4687879.
  28. 28. Crawley O, Barroso C, Testori S, Ferrandiz N, Silva N, Castellano-Pozo M, et al. Cohesin-interacting protein WAPL-1 regulates meiotic chromosome structure and cohesion by antagonizing specific cohesin complexes. eLife. 2016;5. pmid:26841696; PubMed Central PMCID: PMCPMC4758955.
  29. 29. Tedeschi A, Wutz G, Huet S, Jaritz M, Wuensche A, Schirghuber E, et al. Wapl is an essential regulator of chromatin structure and chromosome segregation. Nature. 2013;501(7468):564–8. pmid:23975099.
  30. 30. Luo S, Tong L. Structural biology of the separase-securin complex with crucial roles in chromosome segregation. Curr Opin Struct Biol. 2018;49:114–22. pmid:29452922; PubMed Central PMCID: PMCPMC5915870.
  31. 31. Gutierrez-Caballero C, Cebollero LR, Pendas AM. Shugoshins: from protectors of cohesion to versatile adaptors at the centromere. Trends Genet. 2012;28(7):351–60. pmid:22542109.
  32. 32. Tang TT, Bickel SE, Young LM, Orr-Weaver TL. Maintenance of sister-chromatid cohesion at the centromere by the Drosophila MEI-S332 protein. Genes Dev. 1998;12(24):3843–56. pmid:9869638
  33. 33. Kerrebrock AW, Moore DP, Wu JS, Orr-Weaver TL. Mei-S332, a Drosophila protein required for sister-chromatid cohesion, can localize to meiotic centromere regions. Cell. 1995;83:247–56. pmid:7585942
  34. 34. Yamada T, Tahara E, Kanke M, Kuwata K, Nishiyama T. Drosophila Dalmatian combines sororin and shugoshin roles in establishment and protection of cohesion. EMBO J. 2017;36(11):1513–27. Epub 2017/05/10. pmid:28483815; PubMed Central PMCID: PMCPMC5452020.
  35. 35. Marston AL. Dalmatian: spotting the difference in cohesin protectors. EMBO J. 2017;36(11):1468–70. Epub 2017/05/10. pmid:28483814; PubMed Central PMCID: PMCPMC5452005.
  36. 36. Resnick TD, Satinover DL, MacIsaac F, Stukenberg PT, Earnshaw WC, Orr-Weaver TL, et al. INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila. Dev Cell. 2006;11(1):57–68. pmid:16824953.
  37. 37. Radford SJ, Jang JK, McKim KS. The Chromosomal Passenger Complex is required for Meiotic Acentrosomal Spindle Assembly and Chromosome Bi-orientation. Genetics. 2012;192:417–29. pmid:22865736.
  38. 38. Trivedi P, Stukenberg PT. A Centromere-Signaling Network Underlies the Coordination among Mitotic Events. Trends Biochem Sci. 2016;41(2):160–74. pmid:26705896; PubMed Central PMCID: PMCPMC4733523.
  39. 39. Smurnyy Y, Toms AV, Hickson GR, Eck MJ, Eggert US. Binucleine 2, an isoform-specific inhibitor of Drosophila Aurora B kinase, provides insights into the mechanism of cytokinesis. ACS Chem Biol. 2010;5(11):1015–20. pmid:20804174; PubMed Central PMCID: PMCPMC3039078.
  40. 40. Jang JK, Rahman T, McKim KS. The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes. Mol Biol Cell. 2005;16(10):4684–94. pmid:16055508.
  41. 41. Suijkerbuijk SJ, Vleugel M, Teixeira A, Kops GJ. Integration of Kinase and Phosphatase Activities by BUBR1 Ensures Formation of Stable Kinetochore-Microtubule Attachments. Dev Cell. 2012;23(4):745–55. pmid:23079597.
  42. 42. Elowe S, Hümmer S, Uldschmid A, Li X, Nigg EA. Tension-sensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore microtubule interactions. Genes Dev. 2007;21(17):2205–19. pmid:17785528; PubMed Central PMCID: PMCPMC1950859.
  43. 43. Lénárt P, Petronczki M, Steegmaier M, Di Fiore B, Lipp JJ, Hoffmann M, et al. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1. Curr Biol. 2007;17(4):304–15. pmid:17291761.
  44. 44. Liu D, Davydenko O, Lampson MA. Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing. J Cell Biol. 2012;198(4):491–9. pmid:22908307.
  45. 45. Das A, Shah SJ, Fan B, Paik D, DiSanto DJ, Hinman AM, et al. Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes. Genetics. 2016;202(1):61–75. pmid:26564158; PubMed Central PMCID: PMCPMC4701103.
  46. 46. Conde C, Osswald M, Barbosa J, Moutinho-Santos T, Pinheiro D, Guimaraes S, et al. Drosophila Polo regulates the spindle assembly checkpoint through Mps1-dependent BubR1 phosphorylation. EMBO J. 2013;32(12):1761–77. pmid:23685359; PubMed Central PMCID: PMCPMC3680734.
  47. 47. Matsumura S, Toyoshima F, Nishida E. Polo-like kinase 1 facilitates chromosome alignment during prometaphase through BubR1. J Biol Chem. 2007;282(20):15217–27. pmid:17376779.
  48. 48. Gilliland WD, Wayson SM, Hawley RS. The meiotic defects of mutants in the Drosophila mps1 gene reveal a critical role of Mps1 in the segregation of achiasmate homologs. Curr Biol. 2005;15(7):672–7. pmid:15823541.
  49. 49. Page SL, Hawley RS. c(3)G encodes a Drosophila synaptonemal complex protein. Genes Dev. 2001;15(23):3130–43. pmid:11731477.
  50. 50. Hall JC. Chromosome segregation influenced by two alleles of the meiotic mutant c(3)G in Drosphila melanogaster. Genetics. 1972;71:367–400. pmid:4624918
  51. 51. Takeo S, Lake CM, Morais-de-Sa E, Sunkel CE, Hawley RS. Synaptonemal complex-dependent centromeric clustering and the initiation of synapsis in Drosophila oocytes. Curr Biol. 2011;21(21):1845–51. pmid:22036182.
  52. 52. Tanneti NS, Landy K, Joyce EF, McKim KS. A Pathway for Synapsis Initiation during Zygotene in Drosophila Oocytes. Curr Biol. 2011;21(21):1852–7. pmid:22036181.
  53. 53. Guo Z, Batiha O, Bourouh M, Fifield E, Swan A. Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila. J Cell Sci. 2016;129(3):531–42. pmid:26675236.
  54. 54. Schittenhelm RB, Chaleckis R, Lehner CF. Intrakinetochore localization and essential functional domains of Drosophila Spc105. Embo J. 2009;28(16):2374–86. pmid:19590494.
  55. 55. Moschou PN, Gutierrez-Beltran E, Bozhkov PV, Smertenko A. Separase Promotes Microtubule Polymerization by Activating CENP-E-Related Kinesin Kin7. Dev Cell. 2016;37(4):350–61. pmid:27219063.
  56. 56. Chaurasia S, Lehner CF. Dynamics and control of sister kinetochore behavior during the meiotic divisions in Drosophila spermatocytes. PLoS Genet. 2018;14(5):e1007372. pmid:29734336; PubMed Central PMCID: PMCPMC5957430.
  57. 57. Blattner AC, Chaurasia S, McKee BD, Lehner CF. Separase Is Required for Homolog and Sister Disjunction during Drosophila melanogaster Male Meiosis, but Not for Biorientation of Sister Centromeres. PLoS Genet. 2016;12(4):e1005996. PubMed Central PMCID: PMCPMC4847790. pmid:27120695
  58. 58. Goldstein LS. Kinetochore structure and its role in chromosome orientation during the first meiotic division in male D. melanogaster. Cell. 1981;25(3):591–602. pmid:6793236.
  59. 59. Paliulis LV, Nicklas RB. Kinetochore rearrangement in meiosis II requires attachment to the spindle. Chromosoma. 2005;113(8):440–6. pmid:15711830.
  60. 60. Tournebize R, Andersen SS, Verde F, Doree M, Karsenti E, Hyman AA. Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis. EMBO J. 1997;16(18):5537–49. pmid:9312013; PubMed Central PMCID: PMCPMC1170186.
  61. 61. Posch M, Khoudoli GA, Swift S, King EM, Deluca JG, Swedlow JR. Sds22 regulates aurora B activity and microtubule-kinetochore interactions at mitosis. J Cell Biol. 2010;191(1):61–74. pmid:20921135; PubMed Central PMCID: PMCPMC2953433.
  62. 62. Miller MP, Unal E, Brar GA, Amon A. Meiosis I chromosome segregation is established through regulation of microtubule-kinetochore interactions. eLife. 2012;1:e00117. pmid:23275833; PubMed Central PMCID: PMCPMC3525924.
  63. 63. Afonso O, Matos I, Pereira AJ, Aguiar P, Lampson MA, Maiato H. Feedback control of chromosome separation by a midzone Aurora B gradient. Science. 2014;345(6194):332–6. pmid:24925910.
  64. 64. Vagnarelli P, Hudson DF, Ribeiro SA, Trinkle-Mulcahy L, Spence JM, Lai F, et al. Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis. Nat Cell Biol. 2006;8(10):1133–42. pmid:16998479.
  65. 65. Vanoosthuyse V, Legros P, van der Sar SJ, Yvert G, Toda K, Le Bihan T, et al. CPF-associated phosphatase activity opposes condensin-mediated chromosome condensation. PLoS Genet. 2014;10(6):e1004415. pmid:24945319; PubMed Central PMCID: PMCPMC4063703.
  66. 66. Tada K, Susumu H, Sakuno T, Watanabe Y. Condensin association with histone H2A shapes mitotic chromosomes. Nature. 2011;474(7352):477–83. pmid:21633354.
  67. 67. Daum JR, Potapova TA, Sivakumar S, Daniel JJ, Flynn JN, Rankin S, et al. Cohesion fatigue induces chromatid separation in cells delayed at metaphase. Curr Biol. 2011;21(12):1018–24. Epub 2011/06/11. pmid:21658943; PubMed Central PMCID: PMCPMC3119564.
  68. 68. Sapkota H, Wasiak E, Daum JR, Gorbsky GJ. Multiple determinants and consequences of cohesion fatigue in mammalian cells. Mol Biol Cell. 2018;29(15):1811–24. Epub 2018/05/31. pmid:29846129; PubMed Central PMCID: PMCPMC6085821.
  69. 69. Gorbsky GJ. Cohesion fatigue. Curr Biol. 2013;23(22):R986–R8. Epub 2013/11/23. pmid:24262830.
  70. 70. Yoshida S, Kaido M, Kitajima TS. Inherent Instability of Correct Kinetochore-Microtubule Attachments during Meiosis I in Oocytes. Dev Cell. 2015. pmid:26028219.
  71. 71. Radford SJ, Nguyen AL, Schindler K, McKim KS. The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes. Chromosoma. 2017;126(3):351–64. Epub 2016/11/12. pmid:27837282; PubMed Central PMCID: PMCPMC5426991.
  72. 72. Holubcová Z, Blayney M, Elder K, Schuh M. Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes. Science. 2015;348(6239):1143–7. pmid:26045437; PubMed Central PMCID: PMCPMC4477045.
  73. 73. Jordan PW, Karppinen J, Handel MA. Polo-like kinase is required for synaptonemal complex disassembly and phosphorylation in mouse spermatocytes. J Cell Sci. 2012;125(Pt 21):5061–72. pmid:22854038; PubMed Central PMCID: PMCPMC3533391.
  74. 74. Parra MT, Viera A, Gomez R, Page J, Benavente R, Santos JL, et al. Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I. J Cell Sci. 2004; pmid:14970259 (Pt 7):1221–34.
  75. 75. Gladstone MN, Obeso D, Chuong H, Dawson DS. The synaptonemal complex protein Zip1 promotes bi-orientation of centromeres at meiosis I. PLoS Genet. 2009;5(12):e1000771. pmid:20011112.
  76. 76. Bisig CG, Guiraldelli MF, Kouznetsova A, Scherthan H, Hoog C, Dawson DS, et al. Synaptonemal complex components persist at centromeres and are required for homologous centromere pairing in mouse spermatocytes. PLoS Genet. 2012;8(6):e1002701. pmid:22761579.
  77. 77. Prajapati HK, Agarwal M, Mittal P, Ghosh SK. Evidence of Zip1 Promoting Sister Kinetochore Mono-orientation During Meiosis in Budding Yeast. G3 (Bethesda, Md. 2018. pmid:30254179.
  78. 78. Newman JR, Wolf E, Kim PS. A computationally directed screen identifying interacting coiled coils from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2000;97(24):13203–8. pmid:11087867; PubMed Central PMCID: PMCPMC27203.
  79. 79. Wong J, Nakajima Y, Westermann S, Shang C, Kang JS, Goodner C, et al. A protein interaction map of the mitotic spindle. Mol Biol Cell. 2007;18(10):3800–9. pmid:17634282; PubMed Central PMCID: PMCPMC1995735.
  80. 80. Tachibana-Konwalski K. Cell division: Hold on and let go. Nature. 2015;517(7535):441–2. pmid:25533954.
  81. 81. Sumara I, Vorlaufer E, Stukenberg PT, Kelm O, Redemann N, Nigg EA, et al. The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. Mol Cell. 2002;9(3):515–25. pmid:11931760.
  82. 82. Alexandru G, Uhlmann F, Mechtler K, Poupart MA, Nasmyth K. Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast. Cell. 2001;105(4):459–72. pmid:11371343.
  83. 83. Katis VL, Lipp JJ, Imre R, Bogdanova A, Okaz E, Habermann B, et al. Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis. Dev Cell. 2010;18(3):397–409. pmid:20230747; PubMed Central PMCID: PMCPMC2994640.
  84. 84. Ishiguro T, Tanaka K, Sakuno T, Watanabe Y. Shugoshin-PP2A counteracts casein-kinase-1-dependent cleavage of Rec8 by separase. Nat Cell Biol. 2010;12(5):500–6. pmid:20383139.
  85. 85. Rumpf C, Cipak L, Dudas A, Benko Z, Pozgajova M, Riedel CG, et al. Casein kinase 1 is required for efficient removal of Rec8 during meiosis I. Cell Cycle. 2010;9(13):2657–62. pmid:20581463; PubMed Central PMCID: PMCPMC3083834.
  86. 86. Mirouse V, Formstecher E, Couderc JL. Interaction between Polo and BicD proteins links oocyte determination and meiosis control in Drosophila. Development. 2006;133(20):4005–13. pmid:16971474; PubMed Central PMCID: PMCPMC2810110.
  87. 87. Malmanche N, Owen S, Gegick S, Steffensen S, Tomkiel JE, Sunkel CE. Drosophila BubR1 is essential for meiotic sister-chromatid cohesion and maintenance of synaptonemal complex. Curr Biol. 2007;17(17):1489–97. pmid:17702574.
  88. 88. Attrill H, Falls K, Goodman JL, Millburn GH, Antonazzo G, Rey AJ, et al. FlyBase: establishing a Gene Group resource for Drosophila melanogaster. Nucleic Acids Res. 2015. pmid:26467478.
  89. 89. Lee T, Luo L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron. 1999;22(3):451–61. pmid:10197526.
  90. 90. Sugimura I, Lilly MA. Bruno inhibits the expression of mitotic cyclins during the prophase I meiotic arrest of Drosophila oocytes. Dev Cell. 2006;10(1):127–35. pmid:16399084.
  91. 91. Telley IA, Gaspar I, Ephrussi A, Surrey T. Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo. J Cell Biol. 2012;197(7):887–95. Epub 2012/06/20. pmid:22711698; PubMed Central PMCID: PMCPMC3384421.
  92. 92. Leibfried A, Muller S, Ephrussi A. A Cdc42-regulated actin cytoskeleton mediates Drosophila oocyte polarization. Development. 2013;140(2):362–71. Epub 2012/12/20. pmid:23250210.
  93. 93. Radford SJ, McKim KS. Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes. J Vis Exp. 2016;116(116):e54666. pmid:27842371.
  94. 94. Wu C, Singaram V, McKim KS. mei-38 is required for chromosome segregation during meiosis in Drosophila females. Genetics. 2008;180(1):61–72. pmid:18757915.
  95. 95. Moore DP, Page AW, Tang TT, Kerrebrock AW, Orr-Weaver TL. The cohesion protein MEI-S332 localizes to condensed meiotic and mitotic centromeres until sister chromatids separate. J Cell Biol. 1998;140(5):1003–12. pmid:9490715
  96. 96. Heeger S, Leismann O, Schittenhelm R, Schraidt O, Heidmann S, Lehner CF. Genetic interactions of separase regulatory subunits reveal the diverged Drosophila Cenp-C homolog. Genes Dev. 2005;19(17):2041–53. pmid:16140985.
  97. 97. Szafer-Glusman E, Fuller MT, Giansanti MG. Role of Survivin in cytokinesis revealed by a separation-of-function allele. Mol Biol Cell. 2011;22(20):3779–90. pmid:21865602.
  98. 98. Schittenhelm RB, Heeger S, Althoff F, Walter A, Heidmann S, Mechtler K, et al. Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. Chromosoma. 2007;116(4):385–402. pmid:17333235.
  99. 99. Logarinho E, Sunkel CE. The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes. J Cell Sci. 1998;111 (Pt 19):2897–909. pmid:9730982.