The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin.

Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to bind microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function.

Introduction

Oocytes are specialised cells that undergo meiotic divisions to produce female gametes. Oocytes form the meiotic spindle to segregate chromosomes using mechanisms shared with mitotic cells, but also need to cope with unique challenges to assemble a spindle. The centrosomes are absent during meiotic spindle assembly in the oocytes of most animals, such as humans and Drosophila melanogaster [1], even though the centrosomes are the canonical microtubule organising centres in mitosis. Furthermore, oocytes have an exceptionally large volume of cytoplasm, but need to limit spindle assembly to only the region around the chromosomes and not in other parts of the oocytes. A failure to suppress spindle assembly away from the chromosomes would lead to inefficient use of cellular resources, and most likely lead to aberrant cellular processes. Therefore, crucial proteins for spindle assembly are likely to be activated only around chromosomes but inactivated away from chromosomes.

The spatial regulation of spindle assembly in oocytes is not yet well understood, but one mechanism involving Ran and importin has been well described. As first shown in Xenopus egg extract, chromosome-associated RanGEF, Rcc1, converts Ran-GDP to Ran-GTP. Then Ran-GTP releases spindle assembly factors, such as TPX2, from inhibitory effects of importins to allow microtubule assembly around chromosomes (reviewed in [2]).

In living oocytes, there is mixed evidence for a role for Ran in chromosome-dependent spindle formation. On one hand, there is evidence that expression of dominant negative Ran impairs or delays meiosis I spindle assembly in human and mouse oocytes [3,4]. On the other hand, even when the Ran gradient was disrupted in a mouse oocyte by expressing hyperactive or dominant negative Ran, the oocyte still assembled a single spindle around chromosomes in meiosis I [5]. Similarly, in Drosophila oocytes, expression of dominant-negative or hyperactive Ran induced mild spindle defects in meiosis I but did not produce ectopic spindles [6]. This suggests that oocytes may have other pathways to spatially regulate spindle assembly, in addition to the Ran-importin pathway. The chromosomal passenger complex (CPC), containing Aurora B kinase, was proposed to provide an alternative chromosomal signal independently of Ran [7,8].

Recently, we proposed a new spatial regulation mechanism in Drosophila oocytes, involving the phospho-docking protein 14-3-3. 14-3-3 proteins are a well-conserved family of small, ubiquitous phospho-docking proteins involved in various cellular processes [9]. The 14-3-3 family is conserved across eukaryotes, with at least 7 different isoforms present in the human genome, while yeast, Caenorhabditis elegans and Drosophila melanogaster have 2 each (reviewed in [10,11]). 14-3-3 primarily exists as a dimer, either a homodimer of the same isoform or a heterodimer of two different isoforms.

Most of the described roles of 14-3-3 are through its direct binding to a target protein, with over 200 binding sites reported in the literature [12]. The majority of reported sites are centred on a phosphorylated serine or threonine, while in some rare cases 14-3-3 can bind in the absence of phosphorylation [13]. There are multiple mechanisms through which 14-3-3 can regulate its targets. For example, 14-3-3 can mask localisation motifs to regulate sequestration or shuttling of targets in or between subcellular compartments [14,15]. Some cases have also been reported of the 14-3-3 dimer acting as an adaptor, bringing together two proteins to increase their activity or interaction [16]. Additionally, Yaffe et al [17] propose that 14-3-3 might act as a “molecular anvil” to instigate conformational change in its binding partners. Finally, 14-3-3 might act as a chaperone of unfolded proteins, or facilitate clustering of proteins or complexes.

The importance of 14-3-3 in spindle formation in oocytes has already been demonstrated. 14-3-3 knockdown in Drosophila oocytes leads to defects in spindle bipolarity [18]. Of the two Drosophila isoforms, knockdown of one (14-3-3ε) alone is sufficient to cause these spindle defects in oocytes. Although knockdown of the other isoform (ζ) alone showed no defects, double knockdown with 14-3-3ε resulted in much more severe spindle defects than 14-3-3ε knockdown alone. Similarly to Drosophila, depletion of 14-3-3η in mouse oocytes disrupts spindle assembly and polar body extrusion [19]. We previously identified a microtubule-crosslinking kinesin Ncd as a 14-3-3 target critical for spindle bipolarity in Drosophila oocytes [18]. Ncd and the mammalian orthologue HSET belong to the kinesin-14 family of minus-end directed microtubule motors [20], and both are required to focus the poles of the spindle in oocytes [21,22]. Our previous study proposed the mechanism by which 14-3-3 and Aurora B kinase allow Kinesin-14/Ncd binding of microtubules only near the chromosomes [18]. In the ooplasm away from chromosomes, 14-3-3 binds to Kinesin-14/Ncd at phosphorylated Serine 96, preventing Kinesin-14/Ncd from binding microtubules. In the vicinity of the chromatin, an additional phosphorylation nearby at Serine 94, by the chromatin-bound kinase Aurora B, inhibits 14-3-3 binding of kinesin-14/Ncd. This removes the inhibition, and specifically allows Kinesin-14/Ncd binding to spindle microtubules, promoting proper bipolar spindle formation.

Given the importance of spatial regulation for the oocyte, we ask if 14-3-3 regulates other important spindle-associated proteins in oocytes, and what mechanisms might be involved. By developing a new biochemical method, we identify various microtubule-associated proteins regulated by 14-3-3 in ovary extract. Among these proteins, of particular interest is the chromosomal passenger complex (CPC) subunit Borealin, because the CPC is one of the master regulators of cell division and not known to be regulated by 14-3-3 in any system. We show that Borealin is bound by 14-3-3, and this binding is differentially regulated by two phosphorylations. Mutations in these phospho-sites disrupt Borealin localisation to the spindle equator/centromeres and function in centromere bi-orientation. Collectively, these findings suggest that 14-3-3 plays a central role in a general regulatory system controlling microtubule-associated proteins (MAPs) in oocytes, analogous to the Ran-importin system.

Results

Identification of proteins regulated by 14-3-3 from Drosophila oocytes

We recently proposed a new mechanism of spatial regulation in oocytes, in which Kinesin-14/Ncd is regulated by the combined action of the phospho-docking protein 14-3-3 in the ooplasm and Aurora B kinase on chromosomes [18].

We hypothesise that this mechanism involving 14-3-3 and Aurora B provides a general way to spatially activate many spindle proteins only when near the chromosomes in oocytes. Here we tested this hypothesis by developing a new biochemical method to systematically identify proteins whose ability to bind microtubules is regulated by 14-3-3 in oocytes.

First, we have developed a novel method of purifying microtubule-associated proteins (MAPs) from Drosophila ovaries ( by adapting a previous method for embryos [23]. We took advantage of Drosophila ovaries being mainly made up of mature oocytes (arrested in metaphase I) by volume with a minimal contribution from mitotic cells. A hundred pairs of ovaries were dissected from mature flies and homogenised on ice to depolymerise microtubules. After the resulting lysate was cleared by ultracentrifugation, endogenous tubulin was polymerised by incubating with paclitaxel and GTP at room temperature. Microtubules, together with their associated proteins, were pelleted through a sucrose cushion by ultracentrifugation. The pellet predominantly consists of tubulins and minimal amounts of other proteins ()

Next we used a 14-3-3 inhibitor, R18. R18 is a synthetic peptide with 20 residues that has been shown to competitively bind to the 14-3-3 phospho-docking site with a higher affinity than native substrates [24]. This inhibitor has been shown to bind to various 14-3-3 isoforms, including ε and ζ in vertebrates [24-26]. We expect it can inhibit both 14-3-3 isoforms (ε and ζ) in Drosophila oocytes, as the same isoforms in Drosophila and vertebrates are more similar to each other than between two different isoforms in the same species. To identify MAPs regulated by 14-3-3 in oocytes, soluble ovary extract from wild type was divided into two aliquots. The 14-3-3 inhibitor R18 was added to one, and as a control, water was added to the other. After incubation with paclitaxel and GTP, microtubules and associated proteins were pelleted from both samples.

These two microtubule fractions were then analysed by LC-MS/MS for identification and label-free quantification.

Quantitative mass-spectrometry has identified proteins potentially regulated by 14-3-3

To determine the relative amounts of each protein in the microtubule fraction with or without 14-3-3 inhibition, six pairs of biological replicates were analysed by label-free quantification using LC-MS/MS (). As a result, we have detected 3,564 proteins in ovaries, including 24 out of 32 known spindle MAPs in our dataset. Among them, we were able to quantify 1,504 proteins (42%) in at least 2 out of the 6 experiments (). These proteins were plotted as a volcano plot with fold differences on the X axis and the confidence level (p-value) on the Y axis (). In this plot, proteins on the top right increased their binding to microtubules under 14-3-3 inhibition. In total, 47 proteins were detected in microtubule fractions at a higher abundance in the presence of the 14-3-3 inhibitor than in its absence with a good confidence (defined by p<0.05 and ratio>1.5; ). 14-3-3 is likely to suppress microtubule binding of these proteins in oocytes. Using equivalent criteria, 20 proteins were detected in microtubule fractions at a lower abundance in the presence of the 14-3-3 inhibitor than in its absence ().

It is possible that some identified proteins are not regulated directly by 14-3-3, and instead form a complex with a protein regulated by 14-3-3. To test this possibility, we examined whether any of these 14-3-3 regulated proteins or their orthologues are known to physically interact with each other using STRING database ([27]; Figs ). Among the 47 proteins that increased microtubule binding in the presence of the 14-3-3 inhibitor, we found 48 known protein-protein interactions, which is significantly higher than expected from a random set of 47 proteins (17 interactions; p = 1.2e-09). This is consistent with the possibility that microtubule binding of some proteins is indirectly regulated by 14-3-3 through interactions with other proteins.

Fig 1

Identification of proteins whose microtubule binding is regulated by 14-3-3.

(A) Dissected ovaries were homogenised and cleared by centrifugation to make soluble extract. It was separated into two and a 14-3-3 inhibitor (R18) was added to one. After microtubules are polymerised by addition of Taxol, they are sedimented by centrifugation through sucrose cushion. The pellets containing microtubules and their associated proteins were analysed by label-free quantitative mass-spectrometry. (B) Volcano plot showing the fold changes of the amounts of each protein detected in microtubule fraction in the presence of the 14-3-3 inhibitor in comparison to its absence on the X axis and the significance (p-value) on the Y axis. The red box contains 47 proteins that significantly increased their microtubule binding under 14-3-3 inhibition (the fold change >. 1.5 and the p < 0.05).

One of the proteins that increased binding to microtubules under 14-3-3 inhibition with a high degree and confidence was a known 14-3-3 regulated protein in oocytes, Kinesin-14/Ncd (). In addition, both subunits of centralspindlin (MKlp1/Pav, RacGAP/Tum) increased their binding to microtubules under 14-3-3 inhibition (Figs ). This is consistent with a previous study that shows MKlp1/Pav is directly regulated by 14-3-3 in human mitotic cells [28], although this has not been shown in oocytes. These results confirmed that our new method can successfully identify 14-3-3 regulated proteins. Similarly, three out of the four CPC subunits (Aurora B, Incenp and Borealin) significantly increased microtubule binding under 14-3-3 inhibition, except the smallest subunit Survivin/Deterin (Figs ). The CPC is not yet known to be regulated by 14-3-3 in any organism, and therefore was investigated further.

A broader question is whether 14-3-3 regulates only a few microtubule-associated proteins or many proteins in oocytes. To gain an insight into this question, we tested whether the presence of predicted 14-3-3 binding sites correlated with either an increase or decrease in microtubule binding under 14-3-3 inhibition (). If any changes under 14-3-3 inhibition are purely due to random statistical variations rather than 14-3-3 regulation, the number of proteins which increase or decrease in microtubule binding would be 50:50, regardless of whether proteins can bind 14-3-3. Among 1,504 proteins quantifiable in our experiments, 600 proteins had at least one site predicted at a high confidence to bind 14-3-3. Interestingly, among these 600 proteins, 375 increased (62%) and 225 decreased (38%) microtubule binding under 14-3-3 inhibition, showing proteins with predicted 14-3-3 binding sites are much more likely to increase microtubule binding (a difference of 150 proteins; p<0.001; ). In contrast, among the remaining 904 proteins without predicted 14-3-3 binding sites, 448 increased (49%) and 456 decreased (51%) microtubule binding, which is not significantly different from 50:50 (p = 0.82; ). This suggests that 14-3-3 potentially affects microtubule binding of many proteins in oocytes, and more often suppresses their microtubule binding than promotes it.

In the case of Kinesin-14/Ncd, 14-3-3 binding is regulated by phosphorylation at two sites, and regulating 14-3-3 binding is important for spatially controlling the microtubule binding activity of Kinesin-14/Ncd in oocytes. If 14-3-3 binding to a subset of proteins is regulated by a common mechanism such as phosphorylation by the same kinases, we may see a bias in sequences surrounding 14-3-3 binding sites in addition to the 14-3-3 binding motif. To gain an insight into regulation of 14-3-3 binding, sequences surrounding predicted 14-3-3 binding sites were compared between proteins whose microtubule binding is, or is not, regulated by 14-3-3. All identified proteins were divided into 5 bins according to the fold change in microtubule association under 14-3-3 inhibition. Sequences around predicted 14-3-3 sites on proteins in each bin were pooled together. As the three middle bins are similar, the top 20% (increase under inhibition), the middle 60% and the bottom 20% (decrease under inhibition) were compared (). Serine at -2, serine at 0 and lysine at -1 are significantly overrepresented among the top 20%, while proline at +2 is underrepresented. As previously shown for Kinesin-14/Ncd, Serine at -2 can be phosphorylated by a kinase and this would negatively regulate 14-3-3 binding. Overrepresentation of serine over threonine at 0 (14-3-3 binding site) might also be interesting, as the phosphatase PP2A-B55 is known to prefer phospho-threonine over phospho-serine for dephosphorylation [29].

Two phosphorylations regulate 14-3-3 interaction with the disordered region of Borealin

Our results showed that three out of the four CPC subunits (Aurora B, Incenp and Borealin) significantly increased their quantities in microtubule fractions under 14-3-3 inhibition (). Previous studies from us and others showed that the CPC has crucial roles in oocytes, including spindle microtubule assembly, spindle organisation, and bi-orientation of homologous centromeres [30-35]. The CPC dynamically localises to chromosomes, centromeres and the spindle equator in oocytes [33,36]. However, the CPC has not been reported to be regulated by 14-3-3 in any organism.

Among the CPC subunits, we identified 3 potential sites in Borealin and Incenp that match a consensus 14-3-3 binding sequence ( by bioinformatic analysis [37]. Among them, one site on Borealin (S163) not only matches the 14-3-3 binding consensus, but also shares a high similarity with the 14-3-3 binding sites of Kinesin-14/Ncd and Kinesin-6/MKlp1/Pav (). A distinct feature of these sites is the serine residue at the -2 position (S161) that matches the Aurora B phosphorylation consensus (R/KxS/T). Indeed, both S161 and S163 are phosphorylated in vivo, according to previous phosphoproteomics studies in Drosophila [38]. This potential 14-3-3 binding site (S163) on Borealin is located in a disordered region between the Incenp/Survivin interaction domain and the Borealin dimerisation domain [39,40] ().

To experimentally test whether 14-3-3 can bind Borealin at phosphorylated S163, this disordered region (residues 113–221) was produced with MBP-tag in bacteria (). As a control, the same region was produced with S163 mutated to a non-phosphorylatable alanine residue (S163A). After purification (), MBP-Borealin(113–221) and the S163A mutant were incubated with human PKD2 kinase. PKD2 was used as it is known to efficiently phosphorylate the similar 14-3-3 binding site in Kinesin-14/Ncd in vitro [18]. After the reaction, MBP-Borealin(113–221) and the S163A mutant were incubated with bacterially produced GST-14-3-3ε (). GST-14-3-3ε was then pulled down by glutathione-beads and analysed by western blot using an MBP antibody (). We found that MBP-Borealin(113–221) was efficiently pulled down with GST-14-3-3ε only after treatment with PKD2 (). In contrast, much less of the non-phosphorylatable S163A mutant was pulled down than wild-type Borealin even after PKD2 treatment (Figs and ). This demonstrated that 14-3-3ε binds to Borealin at S163 in a phospho-dependent manner.

Next, we tested whether an additional phosphorylation by Aurora B can prevent Borealin from binding to 14-3-3, as a potential Aurora B phosphorylation site (S161) is located near the 14-3-3 binding site (S163). MBP-Borealin(113–221) was first incubated with PKD2, Aurora B, both kinases or no kinases, and then tested for 14-3-3 binding by pulling down with GST-14-3-3ε. We found that MBP-Borealin(113–221) phosphorylated by both PKD2 and Aurora B bound poorly to 14-3-3ε, much less than when phosphorylated by PKD2 alone (Figs and ). This indicates that additional phosphorylation by Aurora B prevents 14-3-3ε interaction with Borealin already phosphorylated at S163. To confirm that this Aurora B phosphorylation is at S161, a non-phosphorylatable S161A mutation was introduced to MBP-Borealin(113–221). In a 14-3-3ε pulldown assay, Aurora B failed to prevent binding of 14-3-3ε to PKD2-phosphorylated MBP-Borealin(113–221) carrying this S161A mutation (Figs and ). Therefore, the additional Aurora B phosphorylation at S161 prevents binding of 14-3-3ε to Borealin phosphorylated at S163.

Phospho-regulation of 14-3-3 binding is important for efficient Borealin localisation

Our biochemical analysis showed that 14-3-3 binding to Borealin is differentially regulated by two phosphorylations (). To define the in vivo role for this phospho-regulation in oocytes, we generated GFP-tagged versions of two non-phosphorylatable mutants at the 14-3-3 binding site (S163A) and the Aurora B phosphorylation site (S161A).

Their localisation was examined in the absence of the endogenous protein by expressing the GFP-tagged RNAi-resistant wild-type and non-phosphorylatable mutants together with a short hairpin RNA (shRNA) against the endogenous gene, and the chromosomal marker Rcc1-mCherry in oocytes. Live imaging showed that wild-type Borealin localised to the spindle equator and centromeres (), as reported previously for the CPC subunits [33,41].

A non-phosphorylatable Borealin mutant at the 14-3-3 binding site (S163A) nearly abolished the localisation on both the spindle equator and centromeres (). The non-phosphorylatable Borealin mutant at the Aurora B phosphorylation site (S161A) still localised, but with a reduced intensity, on both the spindle equator and centromeres in comparison to wild-type Borealin (). For quantification, a total intensity of GFP signals including both the spindle equator and centromeres above the background were measured. The S163A mutation resulted in a dramatic drop in the GFP signal intensity by 82% in comparison to wild-type Borealin (p<0.001), while the S161A mutation resulted in a less dramatic but significant reduction (by 59%; p<0.001) (). In contrast, intensities of the chromosome signal (Rcc1-mCherry) in the phospho-mutants were not significantly different from those in wild-type Borealin, suggesting that the difference in Borealin signal intensity is due to the reduction in localisation, rather than differences in imaging (). Immunoblotting suggested that these differences in signal intensity are not explained by the expression levels of GFP-Borealin mutant proteins ().

These results revealed that phospho-regulation of 14-3-3 binding to Borealin is important for Borealin localisation in oocytes. The phospho-dependent 14-3-3 binding is crucial for Borealin localisation, while the phosphorylation by Aurora B is less crucial but still important.

14-3-3 binding to Borealin is important for bi-orienting homologous centromeres

To establish the functional significance of the phospho-regulation of 14-3-3 binding in oocytes, we further examined the oocytes expressing the wild-type or mutant Borealin transgene in a background in which the endogenous gene is silenced. We then examined spindle morphology and centromere positions in mostly metaphase I arrested oocytes by immunostaining tubulin combined with in situ hybridisation using a peri-centromere probe specific to chromosome 3 (dodecasatellite; [42]).

We examined oocytes expressing an shRNA against Borealin and without Borealin transgenes alongside a control without shRNA expression (). In the control without shRNA expression, one centromere 3 signal was found on each side of the chromosome mass in nearly all (95%) oocytes, showing a pair of homologous centromeres that are bi-oriented and pulled apart to both poles. Only 5% of oocytes showed a mono-orientation in the control. In contrast, 30% of RNAi oocytes showed mono-orientation (p<0.001). The spindle morphologies look similar between Borealin RNAi oocytes and the control. This is likely to reflect partial depletion of Borealin, although we cannot exclude the possibility that Borealin is not essential for normal spindle morphology.

Expression of GFP-tagged wild-type Borealin fully rescued the centromere bi-orientation defect of Borealin RNAi, and the frequency of mono-orientation went back to a similar level to oocytes without RNAi. In contrast, expression of GFP-tagged Borealin(S163A) failed to rescue the defect, and the frequency of mono-orientation stayed at a similar level to the RNAi oocytes without a transgene. This is consistent with the large reduction in localisation to the spindle/centromeres we observed above (). Expression of GFP-tagged Borealin(S161A) showed an intermediate effect, which is also consistent with partial loss of localisation. These results showed that phosphorylation for 14-3-3 binding at S163 is essential for Borealin function, while phosphorylation of the flanking site (S161) is not essential but appears to play a role. These results showed that phospho-regulation of 14-3-3 binding is important for the centromere bi-orientation function of Borealin.

Discussion

By microtubule co-sedimentation combined with quantitative mass-spectrometry, we identified various proteins whose microtubule binding is regulated by 14-3-3 in Drosophila oocytes. Among them, we showed that 14-3-3 binds to Borealin, a subunit of the chromosomal passenger complex (CPC). Two phosphorylations regulating this 14-3-3 binding to Borealin are important for Borealin localisation and centromere bi-orientation in oocytes.

Using a newly developed method, we quantified the relative amounts of over 1,500 proteins associated with microtubules in ovary extract with or without 14-3-3 inhibition. Our analysis showed that 14-3-3 suppresses microtubule binding of a substantial number of proteins in ovaries. Among these proteins, Kinesin-14/Ncd and the centralspindlin subunits were previously reported to be regulated by 14-3-3 [18,28] in Drosophila oocytes and human cells, respectively. We also identified three subunits of the CPC, one of the master regulators of cell division [32,43]. To our knowledge, this is the first report to show that the CPC is regulated by 14-3-3.

We found that the CPC subunit Borealin has a predicted 14-3-3 binding site (S163) in its disordered middle region. The surrounding sequence shares a striking similarity to the 14-3-3 binding sites identified in Kinesin-14/Ncd and the centralspindlin subunit MKlp1/Pavarotti [18,28]. Through in vitro studies, we showed that 14-3-3ε binds to Borealin at S163 in a phospho-dependent manner, although the kinase that phosphorylates this site in oocytes is unknown. Additional phosphorylation by Aurora B, probably at S161, prevents 14-3-3ε binding, in a similar manner to that reported for Kinesin-14/Ncd [18]. A non-phosphorylatable mutation predicted to abolish 14-3-3 binding (S163A) dramatically reduced the amount of Borealin at both the spindle and centromeres. This mutation also abolishes the function of Borealin in bi-orienting homologous centromeres. On the other hand, a non-phosphorylatable mutation of the predicted Aurora B site (S161A) also compromises the normal localisation and function of Borealin but does not abolish either. This partial requirement of the Aurora B phosphorylation can be explained if 14-3-3 binding to Borealin is prevented by redundant action of two enzymes: a phosphatase dephosphorylating the 14-3-3 binding site (S163) and Aurora B phosphorylating the neighbouring site (S161). Similar effects were also observed in the equivalent mutations in Kinesin-14/Ncd [18], strengthening our hypothesis that a common mechanism regulates Borealin and Kinesin-14/Ncd.

Based on the model proposed by Beaven et al [18] for Kinesin-14/Ncd, we propose that Borealin is prevented from binding microtubules in the oocyte cytoplasm by its association with 14-3-3. When it is in the vicinity of the chromatin, Aurora B kinase activity reverses the 14-3-3 binding and allows the CPC to bind spindle microtubules. We showed that this regulation is important for CPC’s role in bi-orienting homologous centromeres. In our model, spatial regulation mediated by 14-3-3 and Aurora B allows Borealin to bind selectively to spindle microtubules. Abolishing this spatial regulation would dramatically reduce the effective concentration of Borealin near the spindle by binding to numerous non-spindle microtubules in the large volume of the oocyte. This would result in a reduction of Borealin localisation to both centromeres and spindle microtubules. However, we cannot rule out that 14-3-3 and Aurora B may regulate Borealin localisation to both centromeres and spindle microtubules differently from Kinesin-14/Ncd.

This study has established 14-3-3 as a general regulator of microtubule-associated proteins (MAPs). 14-3-3 may play an important role in spatially regulating many MAPs in oocytes in combination with other regulators such as Aurora B or phosphatases, in parallel to the Ran/importin system. Future studies of these 14-3-3 regulated proteins will shed light on how microtubules are regulated in oocytes. The study has also revealed a novel regulation of the CPC. The CPC is one of the conserved master regulators of cell division, and dynamically localises to chromosomes, centromeres and spindle microtubules. Although the chromosome/centromere localisation of the CPC has been intensely studied, microtubule binding and its regulation are paid much less attention. Our study identified a novel mechanism in which 14-3-3 regulates the microtubule binding activity of the CPC. However, the CPC is known to have other microtubule binding domains than this disordered region of Borealin [44] and Incenp may also bind 14-3-3 at the sites we show above. Further studies are required to establish whether this region of Borealin contains a new microtubule binding domain and how multiple microtubule binding sites and 14-3-3 binding sites may work together to regulate the localisation and microtubule binding of the CPC as a whole.

Beyond meiosis, the same mechanism may also operate during cytokinesis when the CPC accumulates to the midbody. As our proposed mechanism can provide a self amplification loop by which the CPC promotes further recruitment of the CPC, in theory this could confer a localised switch-like property to CPC activity during cytokinesis and/or help to accumulate the CPC to a tighter region.

Materials and methods

Fly maturation and generation of GFP-Borr flies

Standard fly techniques were followed [45]. Drosophila melanogaster stocks were cultured on standard cornmeal medium at 25°C. w was used as wild-type. Less than 1 day old female adults were matured for 3 days at 25°C in the presence of males before dissection of ovaries.

To generate Borealin transgenes, the full-length Borr open reading frame was amplified from LD36125 by PCR using PrimeStar (Takara) and cloned between AscI and NotI sites of pENTR (ThermoFisher) using Gibson assembly (HiFi; NEB). To make the gene resistant to RNAi, silent mutations (from GCG GTG TTC to GCC GTC TTT) were introduced by PCR using primers containing the mutations and followed by Gibson assembly. Further mutations (TCC to GCC for S163A; AGT to GCC for S161A) were introduced using the same method. The absence of unwanted mutations were confirmed by Sanger sequencing. These entry clones were recombined with the destination vector φMGW [36] by LR Gateway Clonase II (ThermoFisher).

Transgenic flies expressing RNAi-resistant GFP-Borr (wild-type/S163A/S161A) under the maternal α -tubulin promoter were generated using φC31 integrase-mediated transgenesis at the VK18 site [46] on the second chromosome, performed by BestGene Inc. Each transgene of GFP-Borr or variant was then recombined with another transgene at the attP40 site [47] expressing short hairpin (sh) RNA against borr (HMC04381; [48]; 55942 from Bloomington Drosophila Stock Center).

Microtubule cosedimentation with the 14-3-3 inhibitor R18

Microtubules and their associated MAPs were purified from ovaries by modifying protocols previously used in embryos [49] and in S2 cells [23]. Ovaries from matured female w flies were dissected in 1x BRB80 buffer (80 mM PIPES-KOH pH 6.8, 1 mM MgCl2, 1 mM Na3EGTA) supplemented with phosphatase inhibitors (1mM DTT (Promega), 1mM PMSF, Complete EDTA-free Protease Inhibitor Mixture Tablets MINI, diluted according to manufacturer instructions (Roche), 15 mM Na3VO4, 10 mM p-nitrophenyl phosphate, 1 μM okadaic acid). Ovaries were snap frozen in liquid nitrogen and stored at -80°C. About 100 pairs of ovaries (~150 mg in wet weight) were defrosted and pooled. An equal volume of 2x BRB80+phosphatase inhibitors was added. Ovaries were homogenised in a pre-cooled Dounce homogeniser and incubated on ice for 30 minutes to depolymerise microtubules, then cleared by centrifugation at 13,000 rpm/13,628 x g for 15 minutes at 4°C. The supernatant was cleared again by 2 rounds of ultracentrifugation at 100,000 rpm (358,400 x g; Beckman TLA-120.2) for 10 minutes at 0°C, and then incubated at 30°C for 20 minutes, followed by ultracentrifugation at 100,000 rpm for 10 minutes at 22°C. Actin was depolymerised by the addition of 1 μg/mL Latrunculin A (Cambridge Bioscience), 2 μg/mL Latrunculin B (Cambridge Bioscience), 0.1 mM DTT (Promega) and 1 mM GTP (Sigma), and an aliquot of the lysate was kept for analysis (‘input’).

The remaining supernatant was split into two portions, one of which (‘sample’) was treated with R18 peptide (R18 trifluoroacetate, Sigma) at a final concentration of 100 μM to inhibit 14-3-3 activity. To the other (‘control’) was added an equal volume of water. Both were then incubated for 5 minutes at room temperature. Paclitaxel (Sigma-Aldrich) was subsequently added to both sample and control at a final concentration of 20 μM, and incubated for 30 minutes to allow microtubules to polymerise. Microtubules were pelleted twice through 50% sucrose cushions containing 20 μM paclitaxel via ultracentrifugation at 50,000 x g for 35 minutes at 22°C. Pellets were resuspended in 50 μl of 1x BRB80 + phosphatase inhibitors supplemented with 20 μM paclitaxel. An equal volume of 3x SDS sample buffer + 5% β-mercaptoethanol was added to samples before boiling at 95°C for 2 minutes to denature proteins. Samples were stored at -20°C. Aliquots of samples were analysed by western blot. Total proteins on the membrane were stained with MemCode reversible staining kit (Thermo-Fisher) first, and then with a rat polyclonal antibody against α-tubulin (1:500) followed by IRDye 800CW conjugated goat anti-mouse IgG antibody (LI-COR). The signals were detected with an Odyssey CLx imaging scanner (LI-COR). The brightness and contrast were adjusted uniformly across the entire area in a linear manner without removing or altering features.

Mass spectrometry

Pellets from microtubule cosedimentation were separated using a NuPAGE 12% Bis-Tris gel with MOPS running buffer and stained with Colloidal Blue Staining Kit (Invitrogen). 2 lanes were run for each pellet. The gel region containing the tubulin band was excised and discarded from both sample and control lanes. The remaining gel regions were trypsin-digested and reduced/alkylated using standard procedures [50]. Following digestion, samples were diluted with an equal volume of 0.1% TFA and spun onto StageTips as described by Rappsilber et al. [51]. Peptides were eluted in 80% acetonitrile in 0.1% TFA and concentrated 40x by vacuum centrifugation.

Samples were prepared for LC-MS/MS analysis by diluting them to 5 μL with 0.1% TFA. MS-analysis was performed on an Orbitrap Fusion Lumos tribrid mass spectrometer (Thermo Fisher Scientific, UK) coupled on-line to Ultimate 3000 RSLCnano Systems (Dionex, Thermo Fisher Scientific, UK). Peptides were separated by a PepMap RSLC C18 EasySpray column (2 μm, 100Å, 75 μm x 50 cm) (Thermo Fisher Scientific, UK), operating at 50°C. Parameters are described in . The peptide gradient was: 2 to 40% buffer B in 140 min, then to 95% in 11 min. The percentage of buffer B remained at 95 for 5 minutes and returned back at 2 one minute after. Peptides were selected and fragmented by higher-collisional energy dissociation (HCD) [52] with normalised collision energy of 30. Raw files were processed using MaxQuant software platform [53] version 1.5.2.8 against the complete Drosophila melanogaster proteome (Uniprot, released in September 2016), using Andromeda [54]. Raw mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [55] with the dataset identifier PXD030445.

Bioinformatic analysis

Peptides were assigned to Uniprot accession numbers, and arranged as one accession per row. When more than one accession was assigned per gene, the accession with the lower score was removed. Accessions assigned only one peptide cannot be confidently identified and so were removed. Accessions denoting different RNA transcripts from the same gene were collapsed into one accession, again keeping the highest scored accession in the event of duplicates. The ratio between sample and control intensities for each gene was calculated as log2(sample)–log2(control). When either the sample or control intensity was 0, these accessions were removed from the graph and the statistical analysis and analysed separately.

To determine the consistency between replicates, log2(sample) and log2(control) value distributions were compared between replicates, as well as the line of best fit for the graph of log2(sample) vs log2(control). 6 out of 8 replicates were determined to be similar enough to continue with analysis. To compare the sample and control intensities for each accession, the given accession had to be detected in at least two replicates for both sample and control. The ratios were calculated as above, and the p-value for each ratio calculated using a two-tailed unpaired t-test. Then the ratio and p-value for each protein were plotted in log2 as a volcano plot ().

Gene ontology terms were downloaded from Flybase [56]. Data manipulation and statistical analyses were performed using Excel (Microsoft) and RStudio (RStudio Team, 2020) using the R scripting language [57]. Graphs were plotted using ggplot2 [58]. Candidate 14-3-3 binding sites were predicted using the 14-3-3 Pred tool [37] on the basis of a consensus score above 0.9 and individual scores above the default thresholds (ANN 0.55; PSSM 0.80; SVM 0.25) for all 3 prediction methods. Likely kinase target sequences were predicted in Borealin sequence using the GPS 3.0 programme [59] with medium threshold. Protein fasta sequences were obtained from UniProt. Protein interaction data for network analysis was downloaded from STRING-db [27] on 29 Nov 2021. Included interactions were limited to physical subnetwork above medium confidence (0.400) sourced from experiments and databases. 14-3-3 site sequence comparison analysed and presented using Weblogo (https://weblogo.berkeley.edu/, [60]).

GST-14-3-3 binding to MBP-Borr including phosphorylation

To generate a construct expressing MBP-Borr(113–221) in E. coli, Borr open reading frame corresponding to amino acids 113–221 was first amplified with a stop codon from LD36125 by PCR using PrimeStar (Takara) and cloned between AscI and NotI sites of pENTR (ThermoFisher) using Gibson assembly (HiFi; NEB). The S161A mutation (AGT to GCC) or the S163A mutation (TCC to GCC) was introduced to this entry clone by PCR-amplifying the entire plasmid using primers carrying the mutation followed by Gibson assembly (HiFi; NEB). These entry clones were recombined using LR clonase II (ThermoFisher) with a destination vector modified from pMAL-c2 (NEB) by insertion of the Gateway cassette (ThermoFisher) at the XmnI site. GST-14-3-3ε was produced using a construct previously reported [18].

To test if Borealin is bound by 14-3-3, MBP-Borr(113–221) and GST-14-3-3ε were purified from E. coli (BL21/pLysS) carrying the expression constructs using amylose beads (NEB) and S-glutathione beads (GE Healthcare), respectively. Fractions were eluted using 10 mM reduced glutathione through a polypropylene column (Qiagen), and those with the highest concentrations combined. Combined elutes were then dialysed overnight using Slide-a-Lyser dialysis cassettes (Thermo) in 1 l of dialysis buffer (50 mM Na-phosphate buffer pH7.6, 250 mM KCl, 1 mM MgCl2, 5 mM β-mercaptoethanol). Subsequently, the dialysed purified GST-14-3-3ε was concentrated to 500 μl total using Ultra-4 Spin Columns (Amicon), before being aliquoted, supplemented with 10% final concentration of glycerol, snap frozen in liquid nitrogen and stored at -70°C.

After thawing on ice, 3 μg (3.75 μl) of purified MBP-Borr(113–221) protein (wild-type, S161A and S163A) were cleared of aggregates by centrifugation for 1 minute at 14,000 rpm (13,628 x g), and incubated with 20 μl washed S-glutathione beads for 30 minutes at 4°C. After the beads were removed by centrifugation, 7.5 μg of MBP-Borr(113–221) was incubated with either 450 ng Aurora B (Cambridge Biosciences), 337.5 ng of PKD2 (Cambridge Biosciences), both Aurora B and PKD2 or no kinase. Phosphorylation was carried out in a two-step incubation at 30°C in phosphorylation buffer (20 mM HEPES pH 7.4, 2 mM MgCl2, 1 mM ATP, 27.5 mM KCl, 1 mM DTT, 0.2 mg/mL BSA, 1 mM EGTA), with Aurora B added for the first 30 minutes, followed by addition of PKD2 and a further 60 minutes at 30°C. Phosphorylated fragments (5 μl) were then mixed with 20 μg GST or GST-14-3-3 in 500 μl Pulldown buffer (25 mM Tris-Cl pH = 7.6, 150 mM NaCl, 0.5% Triton X-100, 0.3 mM Na3VO4) and allowed to bind for 30 minutes on ice. They were then incubated with 20 μl washed S-glutathione beads on a rotator at 4°C for 1 hour. The supernatant/unbound fraction was removed and kept for analysis, and beads were then washed 3 times in 800 μl of Pulldown buffer. 2x SDS loading buffer was added to beads and supernatant samples at 1:1 ratio, supplemented with 5% final volume of β-mercaptoethanol, and were boiled for 2 minutes at 95°C, before analysis by western blot. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane using the Mini-PROTEAN and Mini Trans-Blot system (Biorad). Total proteins on the membrane were stained with MemCode reversible staining kit (Thermo-Fisher). After being destained, the membrane was incubated with a rat polyclonal antibody against MBP (1:500) followed by IRDye 800CW conjugated goat anti-rat IgG antibody (LI-COR). The signals were detected with an Odyssey CLx imaging scanner (LI-COR). The brightness and contrast were adjusted uniformly across the entire area in a linear manner without removing or altering features. The band intensities over the background were quantified by the following method. Two rectangular boxes (S and L) were drawn. Box S includes mainly the band of interest, and Box L includes the band and background areas above and below the band. The total signal intensity over the background was calculated by the formula IS − (NS x [IL − IS] / [NL − NS]), where I and N are the total pixel intensity and pixel number in the specified box (S or L), respectively.

Live-imaging of oocytes

To visualise the localisation of GFP-Borr in live oocytes, flies carrying two transgenes that express GFP-tagged Borr (wild-type, S163A or S161A) under the maternal α-tubulin promoter and shRNA against borr under the UASp promoter were crossed with flies carrying GAL4-VP16 (V37; Bloomington Drosophila Stock Centre 7063) and Rcc1-mCherry [36] both under the maternal α-tubulin promoter. Ovaries from 3-day matured flies were dissected one at a time in Halocarbon oil (700; Halocarbon) on a cover slip. Stage 14 oocytes were identified by their morphology, namely a well-formed chorion and long dorsal appendages. Oocytes which had entered the oviduct were discarded along with other stages. These oocytes were observed under a microscope (Axiovert 200M; Zeiss) attached to a spinning disk confocal head (CSU-X1; Yokogawa) controlled by Volocity (PerkinElmer). A Plan-Apochromat objective lens (63x/1.4 numerical aperture) was used with Immersol 518F oil (Zeiss). Images were taken with a Z-slice interval of 0.8 μm and using 40% laser intensity in the red channel, 100% intensity in the green channel. Maximum intensity projections of the Z-stacks are presented as figures and were used for analyses. Images were exported in the TIFF format and signal intensity measurements carried out in ImageJ as followed. The total signal intensities of GFP-Borealin and Rcc1-mChery on the spindle were estimated using the following method. Two areas (S and L) were drawn on the maximum-intensity projection made from Z series of images. Area S includes mainly the central spindle and centromeres or all chromosomes, and Area L includes this area and surrounding region. The total signal intensity over the background was calculated by the formula IS − (NS x [IL − IS] / [NL − NS]), where I and N are the total pixel intensity and pixel number in the specified area (S or L), respectively.

Immunostaining and fluorescence in situ hybridisation

For immunostaining, ovaries were dissected out of mature flies in methanol as previously described [61]. Sonication was used to disrupt remove the chorion and, which can interfere with antibody penetration. Ovaries were sonicated at 38% amplitude (Vibra Cell VCX500; Sonics) for three 1-second pulses and selected for removal of chorion and vitelline membranes. This was repeated by a further one or two times. The resultant oocytes were washed in 1x PBS, and blocked for 1 hour in blocking buffer (10% foetal calf serum in PBS-T) on a rotator. Oocytes were incubated overnight with primary antibodies diluted in blocking buffer. After washing with PBS-T, fluorophore-conjugated secondary antibodies (Alexa 488, 1:250, or Cy3, 1:1000, in PBS-T) along with DAPI (0.2 μg/ml, Sigma) were added, and incubated for 2 hours in the dark. Finally, oocytes were washed in PBS-T and mounted on microscopy slides in glycerol.

For FISH combined with immunostaining in oocytes, stage 14 oocytes were prepared as for immunostaining, and then post-fixed in 8% formaldehyde as described in [62,63]. An oligonucleotide (CCCGTACTGGT)4 for dodecasatellite [42] near centromere 3 was used. 100 pmol of probe was end-labelled with 2 nmol Alexa546-conjugated dUTP (Invitrogen), 16 nmol of unlabelled dTTP (Promega) and 30 units of terminal deoxynucleotidyl transferase (Promega) in 20 μl of transferase buffer at 37°C for one hour. The reaction was halted by incubation at 70°C for 10 minutes, and then remaining free dTTP was removed using a G25 Mini Quick spin column (Roche). 4 μl of this labelled oligonucleotide was added to ovaries in 40 μl of hybridisation buffer (0.1 g/ml dextran sulphate, 50% formamide, 3xSSC) at 30°C overnight. After washing twice in washing buffer (50% formamide, 2x SSC, 0.1% Triton X100) at 30°C for total 30 minutes, oocytes were further washed three times in 2xSSC+0.01% Triton X100 and once in PBS. After blocking in PBS+0.1% Triton containing 10% fetal calf serum, the immunostaining procedure was followed for antibody staining.

These slides were then observed via a confocal microscopy (LSM800 on AxioObserver Z1; Zeiss) using a Plan-Apochromat objective lens (63x/1.4 numerical aperture) with Immersol 518F oil (Zeiss). Images spanning an entire spindle were taken with a Z-slice interval of 0.5 μm and Zoom 2.0 (pixel size 0.1 μm). Maximum intensity projections of the Z-stacks are presented as figures.

Expression of GFP-Borr in ovaries were assessed by western blot using a rabbit anti-GFP antibody (1:100, Invitrogen, A11122) and IRDye 800CW conjugated goat anti-mouse IgG antibody (LI-COR). The signals were detected with an Odyssey CLx imaging scanner (LI-COR). MemCode reversible staining kit (Thermo-Fisher) was used to visualise any proteins. The brightness and contrast were adjusted uniformly across the entire area in a linear manner without removing or altering features.

Microtubule co-sedimentation from Drosophila ovaries.

(A) Microtubules and associated proteins were co-sedimented with or without the 14-3-3 inhibitor R18 from soluble extract of Drosophila ovaries. The original supernatant, wash of the original pellet, and the final pellet used for mass-spectrometry were analysed by western blot using an α-tubulin antibody and total protein staining. (B) The final pellets were run on SDS-PAGE and stained with Coomassie for mass-spectrometry. Nearly all tubulin in the extract was found in the pellet fraction, which predominantly consists of tubulin with minimal amounts of other proteins, regardless of the presence or absence of R18.

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Physical protein-protein interactions known among 20 proteins with significantly lower amounts in the microtubule fraction under 14-3-3 inhibition.

Proteins with the fold change <0.6667 and the p<0.05 are shown. Red and blue indicate proteins with at least one predicted 14-3-3 binding site and without any, respectively. Lines indicate known physical interactions, and they do not have significantly more interactions than expected.

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Protein purification of GST, GST-14-3-3ε, MBP-Borr(113–221) and MBP-Borr(113–221,S163A) from E. coli.

GST (A), GST-14-3-3ε (B) MBP-Borr(113–221) (C), MBP-Borr(113–221,S163A) (D) and MBP-Borr(113–221,S161A) (E) were affinity-purified using columns containing S-glutathione beads or amylose beads and eluted in buffer containing glutathione or maltose, respectively, as described in Materials and Methods. Purification intermediates were analysed by SDS-PAGE and stained with Coomassie.

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Quantification of MBP-Borr in the GST-14-3-3ε pull down in triplicated experiments.

Experiments shown in Fig 3D–3F were triplicated and presented as different coloured bars. The total signal intensities of the MBP-Borealin bands above the background were normalised to that of PKD2-phosphorylated MBP-Borr in GST-14-3-3 pull down in the same experiment. **, * and ns indicate p<0.01, p<0.05 and p>0.05, respectively. (A) MBP-Borealin(113–221) interacts with GST-14-3-3ε in a manner dependent on phosphorylation at S163. (B) An additional phosphorylation by Aurora B prevents PKD2-phosphorylated MBP-Borealin(113–221) from interacting with GST-14-3-3ε. (C) Aurora B cannot prevent interaction between GST-14-3-3ε and PKD2-phosphorylated MBP-Borealin(113–221) with S161A mutation.

Fig 3

14-3-3 binding to the disordered region of Borealin is regulated by two phosphorylations.

(A) Diagram of domain organisation of Borealin and Incenp proteins. Known protein-protein interactions are indicated as double arrows. Three predicted 14-3-3 binding sites (red circles; Borealin S163 and Incenp S163 and S740) are identified among the CPC subunits. Borealin S163 is located within an intrinsically disordered region between domains interacting with the CPC subunits. MT; microtubule. (B) Sequences surrounding predicted 14-3-3 binding sites. The sequence surrounding Borealin has high similarity to Ncd S96 and Pav S744, containing serine at -2 position that is potentially phosphorylated by Aurora B kinase. (C) Testing interaction between the phospho-docking protein 14-3-3 and the disordered region of Borealin (Borr). Purified MBP-tagged Borealin fragment was incubated with or without PKD2, together with BSA. GST-14-3-3ε was added and pulled down by centrifugation using glutathione beads. Proteins bound to the beads and unbound fraction (supernatant) were analysed by western blotting using an MBP antibody. (D) MBP-Borealin(113–221) interacts with GST-14-3-3ε in a manner dependent on phosphorylation at S163. MBP-Borealin(113–221) and MBP-Borealin(113–221) with S163A mutation were incubated with or without human PKD2 kinase before pulldown by glutathione beads coupled with GST-14-3-3ε. A western blot of proteins bound to the glutathione beads and unbound fractions (supernatants) was carried out using an MBP antibody and total protein staining. Quantification and statistical analysis from triplicated experiments are shown in S4 Fig. (E) An additional phosphorylation by Aurora B prevents PKD2-phosphorylated MBP-Borealin(113–221) from interacting with GST-14-3-3ε. Borealin(113–221) was incubated with human PKD2 kinase alone, human Aurora B kinase alone, both kinases or without kinases, and tested for pull down using GST or GST-14-3-3ε. Bound or unbound proteins were analysed by a western blot using an MBP antibody or total protein staining. PKD2-phosphorylated MBP-Borealin(113–221) specifically interacted with GST-14-3-3ε, while MBP-Borealin(113–221) doubly phosphorylated by PKD2 and Aurora B did not. Quantification and statistical analysis from triplicated experiments are shown in S4 Fig. (F) Aurora B cannot prevent interaction between GST-14-3-3ε and PKD2-phosphorylated MBP-Borealin(113–221) with S161A mutation. MBP-Borealin(113–221) and MBP-Borealin(113–221) with S161A mutation were incubated with or without human Aurora B in the presence of human PKD2 kinase before pulldown by glutathione beads coupled with GST-14-3-3ε. A western blot of proteins bound to the glutathione beads and unbound fractions (supernatants) was carried out using an MBP antibody and total protein staining. Quantification and statistical analysis from triplicated experiments are shown in S4 Fig.

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Mass spectrometry intensity data for all proteins in microtubule fractions with and without R18.

This consists of 3 sheets containing all quantifiable proteins, 47 proteins that significantly increased their microtubule binding under 14-3-3 inhibition (the fold change >1.5 and the p< 0.05) and 20 proteins that significantly decreased their microtubule binding under 14-3-3 inhibition (the fold change <0.6667 and the p< 0.05). geneName: Name/symbol of gene as listed on Flybase. Accession.No: Uniprot accession number, as assigned by MaxQuant. logSAM: The average of all Sample (R18-treated) log2 intensity values. logCON: The average of all Control (untreated) log2 intensity values. pairedp.value : The p-value from a paired Student’s T-test. logSAM1: log2 intensity value from Sample (R18-treated) replicate 1. logCON1: log2 intensity value from Control (untreated) replicate 1. logSAM2: log2 intensity value from Sample (R18-treated) replicate 2. logCON2: log2 intensity value from Control (untreated) replicate 2. logSAM5: log2 intensity value from Sample (R18-treated) replicate 5. logCON5: log2 intensity value from Control (untreated) replicate 5. logSAM6: log2 intensity value from Sample (R18-treated) replicate 6. logCON6: log2 intensity value from Control (untreated) replicate 6. logSAM7: log2 intensity value from Sample (R18-treated) replicate 7. logCON7: log2 intensity value from Control (untreated) replicate 7. logSAM8: log2 intensity value from Sample (R18-treated) replicate 8. logCON8: log2 intensity value from Control (untreated) replicate 8. logratio: (the average of log2SAM)–(the average of log2CON)

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Raw mass spectrometry intensity data for replicate 1.

Sheet 1 (“proteinGroups”) lists all protein groups identified in the experiment. Sheet 2 (“CON, REV filtered”) lists groups after filtering (using reverse decoy database, etc). Sheet 3 (“LFQ”) is a summary using data from sheet 2. . Protein IDs: Ensembl or Uniprot Identifiers of a group of proteins with overlapping sequence, i.e. cannot be distinguished from one another with peptides from this experiment. Majority protein IDs: Proteins with at least half the peptide matches of the leading protein in the group. Peptide counts (all): The number of peptides assigned to this protein group. Peptide counts (razor+unique): The number of unique peptides (not shared with other groups) + razor peptides (can be shared with other groups) assigned to each protein in this group. Peptide counts (unique): The number of unique peptides (peptides which only match this group and no other). Fasta headers: The Uniprot fasta headers for the members of this group, containing accession number, name, description and mass. Number of proteins: The number of proteins in this group. Peptides: The total number of peptides assigned to this group. Razor + unique peptides: The number of razor + unique peptides assigned to this group. Unique peptides: The number of unique peptides (only assigned to this group). Peptides CON1 (or CON2… etc): The number of peptides assigned to this group, in the control (untreated). Peptides SAM1 (or SAM2… etc): The number of peptides assigned to this group, in the sample (R18-treated). Razor + unique peptides CON1: The number of razor + unique peptides assigned to this group in the control (untreated). Razor + unique peptides SAM1: The number of razor + unique peptides assigned to this group in the sample (R18-treated). Unique peptides CON1: The number of unique peptides assigned to this group in the control (untreated). Unique peptides SAM1: The number of unique peptides assigned to this group in the sample (R18-treated). Sequence coverage [%]: Percentage of the lead protein sequence covered by peptides. Unique + razor sequence coverage [%]: Percentage of the lead protein sequence covered by unique + razor peptides. Unique sequence coverage [%]: Percentage of the lead protein sequence covered by unique peptides only. Mol. weight [kDa]: Molecular weight of the protein in kDa. Sequence length: Length of the lead protein sequence. Sequence lengths: Lengths of all proteins in the group. Q-value: The ratio of forward to reverse protein hits (a measure of false discovery rate). Score: The score is calculated by MaxQuant using the formula: Sum PEP Score = ∑−log10(PEP), where PEP = Posterior Error Probability. Identification type CON1: Whether this protein group was identified in the control (untreated) by MS/MS or by matching between runs (can increase identification chances). Identification type SAM1: Whether this protein group was identified in the sample (R18-treated) by MS/MS or by matching between runs (can increase identification chances). Sequence coverage CON1 [%]: Percentage of the lead protein sequence covered by peptides in the control (untreated). Sequence coverage SAM1 [%]: Percentage of the lead protein sequence covered by peptides in the sample (R18-treated). Intensity: The summed intensities for all peptides assigned to this group. Intensity CON1: The summed intensities for all peptides assigned to this group in the control (untreated). Intensity SAM1: The summed intensities for all peptides assigned to this group in the sample (R18-treated). LFQ intensity CON1: The normalised intensity for all peptides assigned to this group in the control (untreated). LFQ intensity SAM1: The normalised intensity for all peptides assigned to this group in the sample (R18-treated). MS/MS Count CON1: Peptide spectrum matches in the control (untreated). MS/MS Count SAM1: Peptide spectrum matches in the sample (R18-treated). MS/MS Count: Peptide spectrum matches in the experiment. Only identified by site: If “+”, a modification site alone was used to identify this protein. Reverse: If “+”, this group is likely a false discovery, identified using the reverse decoy database. Potential contaminant: If “+”, this group is a likely contaminant. id: A unique ID for this protein group. Peptide IDs: The IDs of the peptides assigned to this group. Peptide is razor: Shows which peptides are shared with other groups. Mod. peptide IDs: ID to look up these peptides in associated modifications file. Evidence IDs: ID to look up these peptides in associated evidence file. MS/MS IDs: ID to look up these peptides in associated MS/MS summary file. Best MS/MS: IDs of the peptides with the best matching MS/MS spectra for this group. Oxidation (M) site IDs: IDs to look up these peptides in the associated Oxidation data file. Oxidation (M) site positions: Positions of oxidation sites in the lead protein of this group. . A summary of proteins identified in the experiment, after filtering. Accession No: The Uniprot Accession ID for this protein. Peptides CON1 (CON2, C5, etc): The number of peptides assigned to this protein in the control (untreated). Peptides SAM1 (SAM 2, S5, etc): The number of peptides assigned to this protein in the sample (R18-treated). Mol. weight [kDa]: The molecular weight of the protein. Score: The score calculated by MaxQuant using the formula: Sum PEP Score = ∑−log10(PEP), where PEP = Posterior Error Probability. LFQ intensity CON1: The normalised intensity for all peptides assigned to this group in the control (untreated). LFQ intensity SAM1: The normalised intensity for all peptides assigned to this group in the sample (R18-treated). Protein Name: The Uniprot fasta header(s) for this protein group

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Raw mass spectrometry intensity data for replicate 2.

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Raw mass spectrometry intensity data for replicate 5.

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Raw mass spectrometry intensity data for replicate 6.

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Raw mass spectrometry intensity data for replicate 7.

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Raw mass spectrometry intensity data for replicate 8.

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Amino-acid composition around predicted 14-3-3 binding sites.

The raw numbers of each amino acid at each position around predicted 14-3-3 binding sites, which are used to make Fig 2C. Position 7 in the table is the phosphorylation site essential for 14-3-3 binding, which corresponds to position 0 in the Fig 2C. Group 2, 3 and 4 correspond the top 20–40%, 40–60% and 60–80%, respectively.

Fig 2

Bioinformatics analysis.

(A) Physical protein-protein interactions known among 47 proteins that significantly increased their microtubule binding under 14-3-3 inhibition (the fold change >1.5 and p<0.05; the boxed area in Fig 1C). Red and blue indicate proteins with at least one predicted 14-3-3 binding sites and without them, respectively. The lines indicate physical interactions and their thicknesses indicate confidence levels of evidence. (B) The numbers and proportions of proteins with or without predicted 14-3-3 sites that increased or decreased in the microtubule fractions by 14-3-3 inhibition. (C) Sequences around predicted 14-3-3 binding sties among proteins with top 20%, middle 60% and bottom 20% of all detected proteins in the order of the fold change in microtubule fraction in the presence of the 14-3-3 inhibitor. Coloured boxes indicate the residues whose frequencies are significantly different among the top 20% than the frequencies among the rest of proteins. ** indicates p<0.01.

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Quantification of pulldown experiments.

Quantification and statistical analysis of the band intensities from triplicated experiments in Fig 3D–3F.

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Quantification of GFP-Borr signal.

Quantification of GFP-Borealin signal, on which Fig 4B is based on.

Fig 4

Non-phosphorylatable mutations compromises the localisation of Borealin and bi-orientation of centromeres in oocytes.

(A) Non-phosphorylatable mutations (S163A, S161A) reduce the Borealin localisation to the spindle and centromeres. Fluorescence was observed in live oocytes expressing the GFP-tagged RNAi-resistant wild-type or non-phosphorylatable mutants together with a short hairpin RNA (shRNA) against the endogenous gene and a red chromosomal marker, Rcc1-mCherry. The images were presented using the same condition of capture and contrast adjustment for comparison. (B) The total signals of GFP-Borealin and Rcc1-mCherry on the spindle and centromeres above the background was quantified. Red lines indicate the median signal intensities. *** and ** indicate p<0.001 and p<0.01, respectively (Wilcoxon rank sum test). (C) Western blot of ovaries expressing GFP, wild-type ovaries without expression of GFP, and ovaries expressing wild-type or mutant GFP-Borr (S163A or S161A) in the background of borr RNAi, probed with an anti-GFP antibody and protein staining. (D) Non-phosphorylatable mutations (S163A, S161A) compromises the Borealin function in the bi-orientation of centromeres. α-tubulin and peri-centromere 3 (dodecasatellite; arrowheads) were visualised by Immunostaining combined with in situ hybridisation in borr RNAi oocytes with or without expression of the wild-type or non-phosphorylatable Borealin mutants. (E) Frequencies of bi-orientation of homologous centromeres of chromosome 3. Two separate dodecasatelite signals near both ends of the chromosome mass are considered as bi-oriented centromeres, and one or closely located two signals on the one side of the chromosome mass are considered as mono-oriented centromeres. *** and * indicate p<0.001 and p<0.05, respectively (Fisher exact test).

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Frequencies of bi-oriented homologous centromeres of chromosome 3.

The numbers of oocytes showing bi-oriented or mono-oriented homologous centromeres of chromosome 3, on which Fig 4E is based on.

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Parameters used for mass-spectrometry.

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14 Jan 2022

Dear Dr Ohkura,

Thank you very much for submitting your Research Article entitled 'The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin' to PLOS Genetics.

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

All three reviewers were enthusiastic about this work, and in balance, the result is somewhere between a major and minor revision. There are a quite a few comments related to making the paper more accessible by defining terminology or improving the organization. There is also some requests for more quantification and questions regarding the regulation of CPC centromere localization, and the function of S161 and S163. These comments can mostly be addressed by changes to the writing. In your rebuttal, however, also please address the requests for more experiments.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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Reviewer's Responses to Questions

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Reviewer #1: This manuscript by Repton, et.al. addresses the question of how spindles form in oocytes lacking centrosomes, specifically focusing on how the phospho-docking protein 14-3-3 regulates this process. A previous study from this lab demonstrated that 14-3-3 suppresses microtubule binding of Ncd away from chromosomes, to promote acentrosomal spindle assembly. This motivated the authors to search for additional proteins regulated by 14-3-3 in oocytes. They used a mass spectrometry approach to identify proteins whose binding to microtubules is increased/decreased by 14-3-3. The authors went on to study one of these newly identified factors in more detail, demonstrating that 14-3-3 binds to Borealin (a subunit of the CPC), and that two phosphorylations on Borealin regulate this binding. The authors then went on to mutate these sites in vivo, revealing effects on Borealin localization and centromere bi-orientation.

This study addresses an important problem and reports interesting findings that advance the field. The experiments are generally well executed, and the list of 14-3-3 regulated proteins generated will be a good resource for the field. Moreover, the work on Borealin represents a nice combination of in vitro and in vivo work. Despite these strengths, I have some suggestions that I think will improve the paper. Many of these are aimed at making the manuscript accessible to the broad readership of PLoS Genetics. While I think that the readers of this journal will find the topic of interest, the addition of more background information and some reorganization of the supplementary information will make it easier for a broad audience to understand (details below). I think that the below suggestions will improve this interesting paper.

Specific points

1. There is one experiment that I think could better connect the in vitro analysis in Figure 3 with the in vivo analysis in Figure 4. The authors note in the discussion (lines 336-338) that the non-phosphorylatable mutant they analyze in vivo is predicted to abolish 14-3-3 binding. However, this is not directly demonstrated in the manuscript (and doing so would better support their model). I suggest that they use the Borealin S163A (non-phoshorylatable) mutant in the pull-down experiments (same experiment as Figure 3D, but instead of WT Borealin (113-221), use this same fragment but with S163A).

2. The mass spectrometry data is likely to be a useful resource for the field and is a strength of the paper. Because of this, I think it would be helpful for the authors to better organize and present these data, so that readers can more easily analyze it. For example, I was not familiar with some of the terms in the supplementary tables (“LFQ intensity C6”, “LFQ.intensity.SAM”, “logSAM”, how the “score” was determined, etc.) but I could not find legends for these tables, so it was difficult for me to find this information. Also, I couldn’t figure out why the proteins were listed in the order they were – they didn’t appear to be sorted by accession number, or score, or number of peptides…I think it would be helpful to order/sort the lists of proteins in some rational way (maybe by which hits have the most confidence? Or by accession number?), and then provide legends to explain what is displayed in the tables and how the proteins are ordered.

3. Similar to comment #2 above, Table S1 in particular reports data that the field will be interested in. However, I think that it could be better organized. If I understand correctly, this table lists the proteins shown in Figure 1B; some of these proteins increase their binding to microtubules upon 14-3-3 inhibition (right side of the volcano plot), while others decrease (left side). It might be helpful if Table S1 was organized so that readers could easily tell which proteins increased/decreased/stayed the same. This information was provided for each protein in the table, but sorting the list somehow to reflect this value (maybe by ratio?) might help. Finally, I think it might be nice for the authors to make a separate table listing only the 47 proteins highlighted in the results section (lines 170-172); the authors point out that these are the hits with the most confidence, so I think readers might appreciate having a separate table listing only these proteins (rather than searching for these proteins in the larger list in Table S1).

4. The legend for Figure 1 has an error (it says there is a protein gel in Figure 1B, but this is not the case).

5. I was confused by one aspect of the model presented in the discussion, and think that it would be useful for the authors to expand upon and clarify their model so that the readers better understand the take-home message of the paper (perhaps they could also consider adding a model figure?). The authors propose that 14-3-3/Aurora B regulate the ability of Borealin to bind to spindle microtubules (lines 349-350) and that this regulation is important for biorientation (lines 350-351). However, in the presented experiments, it looks like the S163A mutant has reduced binding to both microtubules and centromeres – I don’t see the centromere dots in Figure 4A, and the fluorescence quantified in 4B is the total fluorescence and does not distinguish between microtubule and centromere populations. Given this, I think it possible that 14-3-3 may also regulate the ability of the CPC to bind centromeres. If this is the case, then could it be that delocalization of the CPC from centromeres is what causes the biorientation defects? If so, I think the authors should discuss this possibility – a broader discussion how the authors envision the regulation working would enhance the manuscript.

6. It would be helpful for the reader if there was more background information on Drosophila 14-3-3 proteins. For example, it is noted that there are two isoforms (line 91-92), and that 14-3-3n depletion disrupts spindle assembly (line 107), but more information would be helpful. What is the other isoform called? Have people tried to deplete it, and if so, was there a phenotype? In the authors’ previous study on NCD, did they assess both isoforms? Adding this type of broader background information would help people not familiar with the 14-3-3 literature better understand the current study. Additionally, in the results section the authors mention an inhibitor that they predict inhibits both isoforms. Having more information about the isoforms (e.g. how similar they are) and about the inhibitor would help the reader evaluate this claim.

7. I think it would be helpful to quantify the intensity of the bands on the gels in Figure 3 to give a more quantitative view of how much protein is pulled down. For example, the right-most band in the GST-pull down gel in figure 3D (PKD2 minus) looks a lot weaker than the rightmost band in the GST-pulldown gel in figure 3E (PKD2 minus/AurB minus), even though these are similar experimental conditions. This therefore made me wonder how much the results vary between experiments, so I think doing some sort of quantification of band intensity could help the readers understand the results better. It could be that different amounts were loaded on the gel in the different experiments (the PKD+ band in Figure 3D also looks lighter than the PKD+ band in Figure 3E, so maybe the relative amounts of protein that are pulled down with and without PKD2 are the same in the two experiments); quantifying these experiments would therefore help the reader better interpret the results.

8. This reviewer was left wondering which kinase phosphorylates S163, to mediate the regulation demonstrated in the manuscript. I am not suggesting that the authors do experiments to figure this out (I think that is beyond the score of the manuscript), but I think it would be nice if the authors could speculate on this in their discussion. They use PKD2 in their in vitro experiments, because it had been used previously to phosphorylate a similar site in NCD. But is this the kinase that they think phosphorylates this site in vivo? If not, what are some candidates? This suggestion is optional, but since I think that some other readers might have the same question, I think it would be a nice addition to the paper.

Typos/small wording changes:

- Line 34: “binding” should be changed to “bind”

- Line 66: should be “limit spindle assembly”

- Line 140: should be “ability to bind microtubules”

- Line 148: should be “through a sucrose cushion”

- Line 194: “presence of predicted”

- Line 300: “that are bioriented” (not which)

- Line 302: “Borealin RNAi oocytes” (instead of “RNAi oocytes”)

Reviewer #2: In this manuscript, Repton and colleagues report the identification of a role for 14-3-3e in the regulation of the Chromosomal Passenger Complex (CPC) in meiosis. They show that 14-3-3e binds the CPC subunit Borealin, thereby preventing its interaction with microtubules (MTs). This discovery was enabled by the authors’ development of a new assay for the identification of MT-binding proteins in ovary extracts, which I found impressive. Using 14-3-3 chemical inhibitors, they identified several proteins whose binding to MTs is blocked by 14-3-3. In this way, Ncd was recovered, providing a validation of previous findings by the same group. Borealin was also one of the hits, which they went on to study.

This study makes for a short and compact manuscript that presents few experimental results. Nevertheless, these results are quite novel and significant for the fields of molecular regulation of meiosis and potentially also mitosis. The experiments are sound and generally well controlled, the conclusions are justified by the results, the figures are well presented and the paper is very clearly written. I will be very supportive of publication after consideration of the following points:

1- Do failures in the mechanisms proposed affect egg viability? In the rescue experiments shown in Fig 4D-E, if chromosomes mis-segregate in meiosis, resulting aneuploid embryos may fail to hatch. Only chromosome 3 is scored for bi-orientation. If chromosomes X and 2 were also scored, the percentage of eggs with at least one misoriented chromosome could be very high. However, I don’t know if this experiment would work because the maternal driver used (BDSC 7063) is so strong that it causes a large fraction of embryos to abort, at least in our hands.

2- In Fig 4A-B, the total intensity of GFP-Borr at the spindle + centromeres was measured. However, the mechanism as proposed affects the ability of Borr to bind MTs only. It would be interesting to test if the same mechanism also affects the ability of Borr to localize to centromeres. Could the authors measure the intensity at centromeres only with their existing data?

3- The Western shown in Fig 4C is not clear enough. I think it should be redone. It is important to know that expression levels of the 3 forms of GFP-Borr are equal or very similar. Otherwise, the differences in fluorescence intensity measured could be due to different expression levels.

4- The authors propose that 14-3-3 binding of Borr prevents Borr from binding MTs away from the meiotic spindle. However, this is not tested experimentally. It would be good to test it with the GFP-Borr transgenic flies. To this end, could the authors measure the fluorescence intensity of their different variants of GFP-Borr on MTs of the polar body?

5- In all biochemical experiments, 14-3-3e (epsilon) is used and never 14-3-3z (zeta). Do the authors assume that 14-3-3z would behave in the similar way? This should at least be discussed if not tested. Also, the name 14-3-3e should be used throughout when referring to the protein used in experiments.

6- Results in Fig 3E suggest that 14-3-3e may bind Borr to some degree when unphosphorylated. Would the authors like to comment on it? It looks like this basal binding may be also inhibited by phosphorylation by AurB.

7- Do the authors think that PDK2 may be a kinase that contributes to phosphorylate S163 in vivo? Can they speculate on what other kinases may be involved?

8- I wonder if the regulation of Borr/CPC by 14-3-3 plays a role in cytokinesis. We could imagine that 14-3-3 binding helps prevent CPC binding to the MTs until cytokinesis, when it is recruited to the central spindle. A phosphatase could dephosphorylate S163 during mitotic exit to promote this transition. This may be an idea for future experiments and/or a discussion point.

9- Fig 1B mentioned in the legend (Protein gel) is missing.

Reviewer #3: "The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin" (PGENETICS-D-21-01648) by Prof. Hiroyuki Ohkura and colleagues for PLOS Genetics.

In this paper, the authors used a differential screening approach to identify MAPs regulated by 14-3-3 proteins and required for meiotic spindle assembly. They isolated MAPs and other proteins that show higher or lower affinity for microtubules in the presence of the 14-3-3 inhibitor (R18).

Among isolated proteins they started to study Borealin, a subunit of the Chromosome Passenger Complex. This protein displays a putative phosphorylation site (S163) that matches a 14-3-3 binding site. An Aurora B phosphorylation site is found nearby (S161). This motif is similar to the motif present in the Ncd kinesin. Binding of 14-3-3 to Ncd is regulated by PKD2 and Aurora B Phosphorylation. This team has previously shown an interesting regulatory event for meiotic spindle assembly. PKD2 phosphorylates Ncd on a 14-3-3 binding site. Following phosphorylation, 14-3-3 binds to Ncd that is released from microtubules. However, phosphorylation by Aurora B triggers 14-3-3 release and local binding of Ncd on the meiotic spindle.

They show that the disorder region of Borealin binds to 14-3-3 after adding PKD2. This interaction is inhibited after Aurora B addition. Indicating that this region is likely regulated by both Kinases.

The also show that S161 and S163 are required for centromere and for chromosome bi-orientation.

Main concerns.

I appreciate the differential screening strategy used to isolate proteins (in particular MAPs) sensitive to inhibition by 14-3-3. Riding the wave of their first publication on Ncd, regulated by 14-3-3 and Aurora B, they analyzed the in vitro and in vivo effects of an almost identical motif. Unfortunately, the link between the initial screen for MAPs and the final result on the centromeric de-localization of Borealin are unclear. Finally, several results are correlative and essential controls are missing. The mechanism is speculative.

1-It is essential to show that S161 and S163 of Borealin are essential for interaction with 14-3-3. This has not been shown properly in the in vitro experiments. A S163A mutant should not be able to interact with 14-3-3 in the presence of PKD2. Similarly a S161A mutant should maintain interaction with 14-3-3 in the presence of Aurora B and PKD2. This is essential to connect in vitro and in vivo observations.

Other important concerns that nevertheless need to be amended.

1-This is a matter of taste but I believe too much emphasis is made on the previous Ncd publication. The mechanism involved may be completely different for Borealin.

2- Would it be possible to also include in figure 2 the identity of the proteins that are less present in the MT pellet in the presence of the R18 inhibitor.

3) L611. Where is the gel in the figure 2.

4) RCC1 levels are not comparable and significantly different (there is a discrepancy between Lane 283 and Figure 4B lower panel). Is it because of the image acquisition? Is it because the S161 mutant triggers loss of RCC1 targeting?

5) The images of IF acquisitions are very small and it is difficult to see the differences. Scale bar are missing in some panels.

6) Western Blots are not of optimal quality. Load same amounts of proteins (Figure 4C)

7) Figure 3. Please provide quantitation, I do not see differences in Fig 3D between ++ and +. Alternatively, use one condition.

8) Figure 3 E. There is more MBP-Bor bound to GST-14-3-3 in the presence of PkD2 (well ° 5) but there is obviously more GST14-3-3 on the beads. See previous comment (well 8) There seems to be more bound MBP-Bor to GST-13-3-3 without any of the 2 Kinases, how it is possible?

9) What is the evidence that regulation of interactions between 14-3-3 and Borealin (by PKD2 and Aur B) is regulated via Ser161 and S163. Are these sites phosphorylated in vivo? (See my main concern N°1).

10-There is also a 14-3-3 binding site in INCENP. Please comment for potential combinational effects on CPC localization.

11-L667. Arrowheads are not visible on the figure.

12-In a previous study this team has shown that the CPC regulates MT nucleation on the meiotic spindle. However, MTs appear to be well nucleated in the absence of Borealin that is a member of the CPC complex. This is should be clarified.

13-Usually, the RNAi/Rescue system is validated by Western Blot to challenge depletion of the endogenous protein and persistence of the rescuing tagged protein. This is not shown in this study. Please provide evidence the system is working. Is there a Borealin antibody to validate the strategy?

14-There is confusion between spindle and centromere localization. The screen aimed to find proteins for which 14-3-3 binding abolishes interaction with meiotic MTs. However the Borealin protein (and possibly other CPC subunits) is located on the centromeres and not on the meiotic spindle.

15-Lane 365-366-“ Our study identified a novel mechanism 
regulating microtubule binding activity of the CPC, which is essential for bi-orienting 
homologous chromosomes in oocytes. 
 » There are no direct evidences that MT binding activity of Bor is involved in CPC centromere localization (S161 and S163 appears important). This is over interpretation.

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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14 Mar 2022

Submitted filename: revision letter Repton.pdf

Click here for additional data file.

4 Apr 2022

Dear Dr Ohkura,

Thank you very much for submitting your Research Article entitled 'The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript

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Two of the three reviewers had no or only minor comments. One reviewer requested an additional experiment with the S161A mutation. While it would be an improvement to have this data, I understand that time and technical constraints may make this difficult to accomplish. In addition, the S163A experiment does make the paper stronger. I would like you to consider doing this experiment, but if not, state explicitly (like on pg 11) that you have not shown S161 regulates interactions with 14-3-3 in an AuroraB-dependent manner. I would also like you to emphasize on pg 12 that the Borealin RNAi is probably a partial knockdown, because its phenotype is much milder that the loss of other CPC components. With these changes and the others asked for by two reviewers, I don't think sending the manuscript out for review will be necessary.

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The manuscript by Repton et.al. is substantially improved and the authors did a good job addressing my concerns. The manuscript is now more accessible to a broad audience, and I think will be of interest to the readers of PLOS Genetics.

I found one small typo but otherwise have no further suggestions:

Line 204: “which increases or decreases” should be “that increase or decrease”

Reviewer #2: My points have been addressed in an acceptable manner. I am in favor of accepting the revised manuscript for publication.

Reviewer #3: I had requested a number of additional experiments. Despite the changes in the manuscript, I note that the authors did not perform any of these experiments. This is detrimental to the quality of the manuscript and I think that the in vitro experiment with the S161A mutant would have really provided the missing piece of the puzzle to make a solid story.

I believe that the regulation of mitotic proteins, including CPC by 14-3-3 proteins is an important finding in the field of cell division. However, while I appreciate the changes in the writing that have improved the manuscript, I still have serious concerns because none of the experiments I requested have been performed.

It is really unfortunate that the authors ignored my comment on serine 161. Even if the effect of Aurora B on 14-3-3 is “likely” not due, (according to them,) to another phosphorylation this is not demonstrated. It is all the more unfortunate that the most complicated experiment (in vivo study) was performed with the variant harbouring the S161A mutation. Given the team expertise expertise, I find it hard to believe that the time constraint is the first reason given for not doing this experiment (request for extension period over the 2 months period is usually given by the editors). In the end, it is rather unfortunate for the overall quality of this manuscript because Figure 3 shows the importance of Aurora B in vitro and Figure 4 shows the importance of Ser161 in vivo. There is a real gap here that could have been filled by doing the requested experiment. I suggest doing this experiment, and correcting their protein loading problems in the gels.

Other minor comments (version with annotated changes).

If the experiment in Fig 3 was performed 3 times, there should be some quantification with appropriate tests (n=3, P<, SD…etc).

. “S2 Fig). Among the 47 
proteins that increased microtubule binding in the presence of the 14-3-3 inhibitor, we found 48 known protein-protein interactions among these 47 proteins, which is significantly higher than 
expected from a random set of 47 proteins (17 interactions; p=1.2e-09) » How this is possible ?

. Lane 308. The authors are entangled with their signal quantifications, (Western Blots and IF images). Please clarify. The authors claim The RCC1 levels are not different (this is not reflected by the statistical tests). I understand that making new experiments at this stage using another DNA probe is complicated. I would suggest to use RCC1 as a probe to position the meiotic spindle and to stick on GFP signal quantification. However, this is possible only if the proteins are expressed are similar levels and if the GFP blot is OK. As I mentioned in my previous review the anti-GFP Western blot is far from optimal, I suggest using another GFP antibody, (or performing IP using GFP TRAP beads) and load similar amounts of proteins. It is not an unjustified request to ask for quality Western blots for a high-quality journal like PLOS Genetics.

. Lane 409-411. As our proposed mechanism can provide a self amplification 
loop, in theory this could confer a localised switch-like property to CPC activity during 
cytokinesis and/or help to accumulate the CPC to a tighter region.

I fail to understand the self-amplification loop mechanism, please explain.

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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22 Apr 2022

Submitted filename: revision letter Rev2.pdf

Click here for additional data file.

27 Apr 2022

Dear Dr Ohkura,

We are pleased to inform you that your manuscript entitled "The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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31 May 2022

PGENETICS-D-21-01648R2

The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

Dear Dr Ohkura,

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