The cells in our body are a hive of activity, but that activity must be kept under control. This is never more critical than when a cell divides, because unchecked cell division can lead to cancer. Fortunately, enzymes called kinases and phosphatases exist to control the countless proteins in a cell; these enzymes help ensure that each step of cell division is complete before moving on to the next.

Kinases control other proteins by adding bulky phosphate groups to them, while phosphatases remove those groups. For a long time, phosphatases were assumed to be less specific than their kinase counterparts. Yet it has now become clear that phosphatases achieve specificity by interacting with a range of regulatory subunits.

A phosphatase called PP2A oversees a number of key steps in cell division by working together with its regulatory B56 subunit. In human cells, there are five separate genes that encode B56 subunits, and all of these B56 ‘isoforms’ were thought to exert the same influence on the PP2A phosphatase. The fact, however, that different isoforms are found at different locations within the cell suggested otherwise.

To investigate this, Vallardi et al. focused on a particular stage of cell division when the activity of the PP2A-B56 complex is essential. Before a cell divides it duplicates its genetic material and the two copies of each chromosome are held together until the cell is ready to pull them apart. The experiments compared two representative B56 isoforms: one that concentrates at the centromere, the region where the copied chromosomes are held together; and another found at the kinetochore, a nearby structure that is involved in pulling the two chromosomes apart. By eliminating all but one isoform and measuring the ensuing activity of the PP2A-B56 complex, Vallardi et al. could differentiate between the main regulatory roles of each isoform. These experiments showed that B56 isoforms control separate processes during cell division, which mirrors their different locations within the cell.

Next, Vallardi et al. looked at the receptor proteins that recruit each isoform to its position. Removing or relocating different receptors showed how they anchor select B56 isoforms in different positions while the associated PP2A enzymes get to work on different processes. Further experiments using ‘hybrid’ subunits made from parts of two different B56 isoforms then helped to reveal the site on the B56 subunits that determines which receptors they bind to. Together these findings show that slight differences between each B56 isoform ultimately dictate where they localise and what processes they control when cells divide.

Protein Phosphatase 2A (PP2A) is a major class of serine/threonine phosphatase that is composed of a catalytic (C), scaffold (A) and regulatory (B) subunit. Substrate specificity is mediated by the regulatory B subunits, which can be subdivided into four structurally distinct families: B (B55), B’ (B56), B’ (PR72) and B’’ (Striatin) (Seshacharyulu et al., 2013).

In humans, the B subunits are encoded by a total of 15 separate genes which give rise to at least 26 different transcripts and splice variants; therefore, each of the four B subfamilies are composed of multiple different isoforms (Seshacharyulu et al., 2013). Although these isoforms are thought to have evolved to enhance PP2A specificity, there is still no direct evidence that isoforms of the same subfamily can regulate specific pathways or processes. Perhaps the best indirect evidence that they can comes from the observation that B56 isoforms localise differently during mitosis (Bastos et al., 2014; Nijenhuis et al., 2014). However, even in these cases, it is still unclear how this differential localisation is achieved or why it is needed.

We addressed this problem by focussing on prometaphase, a stage in mitosis when PP2A activity is essential to regulate sister chromatid cohesion (Kitajima et al., 2006; Riedel et al., 2006; Tang et al., 2006), kinetochore-microtubule attachments (Foley et al., 2011; Kruse et al., 2013; Suijkerbuijk et al., 2012; Xu et al., 2013) and the spindle assembly checkpoint (Espert et al., 2014; Nijenhuis et al., 2014). Crucially, all of these mitotic functions are controlled by PP2A-B56 complexes that localise to either the centromere or the kinetochore.

The kinetochore is a multiprotein complex that assembles on centromeres to allow their physical attachment to microtubules. This attachment process is stochastic and error-prone, and therefore it is safeguarded by two key regulatory processes: the spindle assembly checkpoint (SAC) and kinetochore-microtubule error-correction. The SAC preserves the mitotic state until all kinetochores have been correctly attached to microtubules, whereas the error-correction machinery removes any faulty microtubule attachments that may form. The kinase Aurora B is critical for both processes because it phosphorylates the kinetochore-microtubule interface to destabilise incorrectly attached microtubules and it reinforces the SAC, in part by antagonising Knl1-PP1, a kinetochore phosphatase complex needed for SAC silencing (Saurin, 2018). These two principal functions of Aurora B are antagonised by PP2A-B56, which localises to the Knl1 complex at the outer kinetochore by binding directly to BubR1 (Foley et al., 2011; Kruse et al., 2013; Suijkerbuijk et al., 2012; Xu et al., 2013). This interaction is mediated by the B56 subunit, which interacts with a phosphorylated LxxIxE motif within the kinetochore attachment regulatory domain (KARD) of BubR1 (Wang et al., 2016a; Wang et al., 2016b).

As well as localising to the outer kinetochore, PP2A-B56 also localises to the centromere by binding to shugoshin 1 and 2 (Sgo1/Sgo2) (Kitajima et al., 2006; Riedel et al., 2006; Rivera et al., 2012; Tang et al., 2006; Tanno et al., 2010; Xu et al., 2009). The crystal structure of Sgo1 bound to PP2A-B56 has been solved to reveal a bipartite interaction between Sgo1 and the regulatory and catalytic subunits of the PP2A-B56 complex (Xu et al., 2009). This interaction is thought to allow centromere-localised PP2A-B56 to counteract various kinases, such as Aurora B, which remove cohesin rings from chromosome arms during early mitosis in higher eukaryotes (Marston, 2015). The result is that cohesin is specifically preserved at the centromere where it is needed to resist the pulling forces exerted by microtubules. As well as preserving cohesion at the centromere, PP2A-B56 is also thought to balance the net level of Aurora B activation in this region (Meppelink et al., 2015).

In human cells, B56 isoforms are encoded by five separate genes (B56α, β, γ, δ and ε). The interaction interfaces involved in BubR1 and Sgo1 binding are extremely well conserved between all of these B56 isoforms (Figure 1—figure supplement 1). This explains why BubR1 and Sgo1 appear to display no specificity for individual B56 isoforms (Kitajima et al., 2006; Kruse et al., 2013; Riedel et al., 2006; Xu et al., 2013; Xu et al., 2009), and why these isoforms have been proposed to function redundantly at kinetochores during mitosis (Foley et al., 2011).

However, one crucial observation throws doubt over this issue of redundancy: individual B56 isoforms localise differentially to either the kinetochore or centromere in human cells (Meppelink et al., 2015; Nijenhuis et al., 2014). It is therefore not easy to reconcile this differential localisation with the evidence presented above, which implies that the centromere and kinetochore receptors for B56 do not display any selectivity for individual isoforms. This caused us to readdress the question of redundancy and isoform specificity in human cells.

This work demonstrates how different B56 isoforms localise to discrete subcellular compartments to control separate processes during mitosis. Differential B56 isoform localisation has previously been observed in interphase (McCright et al., 1996) and during the later stages of mitosis (Bastos et al., 2014), which implies that B56 isoforms may have evolved to carry out specific functions, at least in part, by targeting PP2A to distinct subcellular compartments. The differential localisation we observe during prometaphase arises because B56 isoforms display selectivity for specific receptors at the centromere and kinetochore.

The centromeric isoform B56α binds preferentially to Sgo2 via a C-terminal stretch that lies between amino acids 405 and 453 (Figure 6—figure supplement 1). A key loop within this region juxtaposes the catalytic domain and contains an important EPVA signature that is critical for Sgo2 binding and is unique to B56α and B56ε. This sequence is also conserved in Xenopus B56ε, which has previously been shown to selectively bind to Sgo2, when compared to B56γ (Rivera et al., 2012). We therefore propose that a subset of B56 isoforms (B56α and ε) utilize unique motifs to interact with Sgo2 and the centromere during mitosis.

How then can these results be reconciled with the fact that Sgo1 appears to be more important than Sgo2 for the maintenance of cohesion during mitosis (Huang et al., 2007; Kitajima et al., 2005; Kitajima et al., 2006; Llano et al., 2008; McGuinness et al., 2005; Rivera et al., 2012; Tang et al., 2004; Tang et al., 2006; Tanno et al., 2010)? Firstly, it is important to note that Sgo1 can compete with the cohesin release factor, WAPL, for cohesin binding (Hara et al., 2014), thereby protecting cohesion independently of PP2A. In addition, Sgo1 could help cells to tolerate the loss of Sgo2, because Sgo2 depletion does not fully remove PP2A-B56 from the centromere, and the pool that remains under these conditions is dependent on Sgo1 (Figure 2a–g). Therefore, the residual Sgo1-PP2A-B56α/ε that remains at centromeres following Sgo2 depletion could be sufficient to preserve cohesion. Finally, Sgo1 is needed to preserve Sgo2-PP2A-B56 at the centromere (Figure 2e) and it can also bind directly to the SA2–Scc1 complex (Hara et al., 2014; Liu et al., 2013b; Tanno et al., 2010). Therefore, perhaps Sgo1 also helps to position Sgo2-PP2A-B56 so that it can dephosphorylate nearby residues within the cohesin complex. It will be important in future to examine the interplay between Sgo1, Sgo2 and PP2A-B56 at centromeres.

The kinetochore B56 isoforms bind to BubR1 via a canonical LxxIxE motif within the KARD (Hertz et al., 2016; Kruse et al., 2013; Suijkerbuijk et al., 2012; Xu et al., 2013). Although the LxxIxE binding pocket is completely conserved in all B56 isoforms (Figure 1—figure supplement 1), we observe a striking preference in the binding of B56γ over B56α to many LxxIxE containing proteins during prometaphase (Figure 4). We hypothesise that this is due to repressed binding between LxxIxE motifs and B56α during prometaphase, because LxxIxE binding (Figure 6e,f) and kinetochore accumulation (Figure 5h,j) can both be enhanced by mutation of the EPVA loop in B56α (B56α^TKHG). We cannot, however, exclude the possibility that the corresponding TKHG sequence in B56γ positively regulates LxxIxE interaction and kinetochore localisation. Considering that this region also controls Sgo2 and centromere binding, a simple explanation could be that Sgo2 interaction obscures the LxxIxE binding pocket. However, this appears unlikely for four reasons: 1) Sgo2 depletion does not relocalise B56α to kinetochores (Figure 2a,b), 2) Sgo2 depletion does not enhance the ability of B56α to bind to BubR1 or other LxxIxE motifs during mitosis (Figure 6—figure supplement 2), 3) centromere and kinetochore binding can occur together in certain B56α-γ chimaeras (Figure 6—figure supplement 1b), and 4) the regions that define each of these localisations do not fully overlap (Figure 6g). Although we believe these results imply that Sgo2 is unlikely to block LxxIxE interaction, in vitro experiments with purified components would ultimately be needed to formally rule this out. If not Sgo2, then what could limit the kinetochore accumulation of B56α? We speculate that another interacting partner, or alternatively a tail region within a PP2A-B56 subunit, might obscure or modify the conformation of the LxxIxE binding pocket in PP2A-B56α complexes.

An important additional finding of this work is that Sgo1 contributes to the B56γ signal observed at the kinetochore (Figure 3g–j). This likely requires Sgo1 to be bound to histone tails, because it also depends on Bub1, the kinase that phosphorylates histone H2A to recruit Sgo1 (Baron et al., 2016; Kawashima et al., 2010; Kitajima et al., 2005; Liu et al., 2013a; Tang et al., 2004; Yamagishi et al., 2010) (Figure 3—figure supplement 1). It is not currently clear whether a Sgo1:PP2A-B56γ complex simply contributes to the signal observed at kinetochores or whether it may help to physically retain BubR1:PP2A-B56 at the kinetochore, for example, by directly interacting with the BubR1:PP2A-B56 complex. The interfaces between BubR1-B56 and Sgo1-PP2A-B56 do not appear to be overlapping, at least based on current structural data (Wang et al., 2016a; Wang et al., 2016b; Xu et al., 2009), which implies that Knl1-bound BubR1-B56 could potentially be anchored towards histone tails by Sgo1. We were unable to detect Sgo1 in YFP-BubR1 immunoprecipitates (results not shown), however, this could simply reflect an interaction that is either transient or unstable away from kinetochores. It will be important in future to clarify exactly how Sgo1 collaborates with BubR1 to control B56 localisation and, in particular, to determine whether Sgo1 can interact with BubR1:PP2A-B56 complexes directly. If such a complex can exist, then this could have important implications for SAC signalling and tension-sensing.

In summary, the work presented here explains how different members of the PP2A-B56 family function during the same stage of mitosis to control different biological processes. This is the first time that such sub-functionalisation has been demonstrated between isoforms of the same B family. It is currently unclear why such specialisation is necessary or at least preferable to a situation whereby all B56 isoforms operate redundantly, as initially suggested (Foley et al., 2011). One possibility is that the use of different B56 isoforms allows PP2A catalytic activity to be regulated differently in specific subcellular compartments: for example, by enabling interactions or post-translational modifications that are specific for the B56 subunits. In this respect, protein inhibitors of PP2A-B56 have been shown to function specifically at the centromere (SET (Chambon et al., 2013)) and at the kinetochore (BOD1 (Porter et al., 2013)); therefore, it would be interesting to test whether these inhibitors display selectivity for certain PP2A-B56 isoforms. Future studies such as this, which build upon the work presented here, may ultimately help to reveal novel ways to modulate the activity of specific PP2A-B56 complexes. The recent development of selective inhibitors of related PP1 regulatory isoforms to combat neurodegenerative diseases (Das et al., 2015; Krzyzosiak et al., 2018), provides a proof-of-concept that successful targeting of specific serine/threonine phosphatase isoforms is both achievable and therapeutically valuable.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures that contain graphical information, which is every figure except Figure 1—figure supplements 1 and 2, and Figure 6 - figure supplement 2

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Thank you for submitting your article "Division of labour between PP2A-B56 isoforms at the centromere and kinetochore" for consideration by eLife. Your article has been reviewed by Andrea Musacchio as the Senior Editor and Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

PP2A occurs mostly as a trimeric enzyme, consisting of a catalytic C-subunit, a scaffolding A-subunit and a regulatory B-subunit. The literature describes four types of structurally unrelated B subunits and their diversity is further augmented by the expression of isoforms. Five different genes encode for the B' (B56) isoform of PP2A. They are highly similar in amino acid sequence suggesting that they share redundant functions. Yet, recent studies in human cells revealed that individual B56 isoforms display distinct localization patterns, i.e. they localize to either the kinetochore or the centromere. In the submitted manuscript, Vallardi et al., investigate the molecular mechanisms underlying this differential localisation and function. They identify a domain in B56 that is responsible for this and show that it modulates the interaction with proteins containing a short linear motif (SLiM). This is probably the most novel observation reported here. Other observations, for instance the role of Sgo2 as the main receptor for centromeric PP2A-B56, are known from previous work (Kitajima et al., 2006), but the present study is systematic and provides a more comprehensive picture. Collectively, the work was praised for being systematic and because it sheds light on the molecular basis of a poorly understood phenomenon. While the analysis is generally strong, a major concern and several other points were discussed.

Essential revisions:

A point emerged strongly in the post-review discussion, namely that quantitative evidence corroborating claims on the role of the newly identified sequence motifs would not simply be nice to have but would strongly reinforce the paper. An analysis in vitro with purified B56a and B56g and peptides containing the relevant sequence motifs should aim to assess whether Sgo2 reduces the binding of B56a to BubR1, whether Sgo1 binds directly to Bubr1-B56, and whether these interactions are affected by mutation of the EPVA or TKHG motifs. Based on a significant body of previous work, we believe that these experiments are feasible and can be carried out in a time compatible with an eLife revision.

Other important points:

For some intensity plots, the authors indicate if the observed differences are significant or not (Figure 2, Figure 3B and D, Figure 2—figure supplement 1, Figure 3—figure supplement 2, Figure 6) but for others they do not (Figure 3H and J, Figure 3—figure supplement 1). Please add this information to all the plots.

In some of the presented images, a clear-cut centromeric or kinetochore localization of B5a and B56g, respectively, as compared to the marker proteins Sgo2 and CENP-C, was not immediately recognisable (see for example Figure 1B). This is particularly true for endogenously tagged B56g, which is almost equally abundant at kinetochores and centromeres (see Figure 1—figure supplement 3D). At the very least, the partially overlapping localisation should be acknowledged.

Kinetochore associated B56g, but not centromeric B56a, was sufficient to maintain the kinetochore functions of PP2A-B56 after the knockdown of all other B56 isoforms. The authors should explicitly state here that this cannot be explained by a lower expression level of B56a in these cells, as is only shown later in Figure 4A (but never commented upon). The reverse experiment, addressing whether B56a alone, but not B56g, can maintain the centromeric functions of PP2A-B56 is lacking and the authors could elect to carry it out.

The relative kinetochore intensity (e.g. Figure 1—figure supplement 5C) apparently refers to the combined kinetochore + centromere targeting. The normalised kinetochore intensity then shows the distribution between kinetochores and centromeres (e.g. Figure 1—figure supplement 5D). The y-axes should be re-defined to make this difference clear.

Figure 1—figure supplement 2. Please include the WB for the other B56 isoforms.

In Figure 2 and Figure 2—figure supplement 1Figure, the authors aim to demonstrate that specific binding of B56a to Sgo2 contributes to its centromeric localization. With these figures, the following problems were identified:

Figure 2C: Upon Sgo1 depletion, B56a seems to change its localization from a primarily centromeric localization to a primarily kt localization. Yet, this is not reflected in the quantifications shown in Figure 2D and 2E. Furthermore, Figure 2D gives the impression that Sgo1 is very efficiently depleted in Sgo1RNAi cells. The IF image shown in C gives a different impression. Could the authors please comment on this?

Subsection “Sgo2 provides specificity for centromeric B56 recruitment”: "…, CB-Sgo2 was only able to recruit B56a (Figure 2h-k)." This statement is puzzling. Figure 2J shows hardly any YFP-B56g localization. Why is the localization so different compared to Figure 1 and how was the quantification based on such IF images performed? Importantly, it should be clarified whether this figure and the related Figure 2—figure supplement 1 are carried out under conditions of depletion of Sgo1 and/or Sgo2, as this does not seem to be stated anywhere.

Subsection “Sgo2 provides specificity for centromeric B56 recruitment”: "…CB-Sgo1 was able to localize both B56a and B56g to the inner kinetochore (Figure 2—figure supplement 1)." This statement is not supported by the data. Under wt conditions, B56g already colocalizes with CENP-C as shown in Figure 1A and 1B. Why are the quantifications (Figure 2K and Figure 2—figure supplement 1D) so different for control conditions?

And given that B56g colocalizes with CENP-C under wt conditions, how do the authors come to the conclusion that CB-Sgo1 recruits B56g to inner kts?

5) Along the same line, B56a shows a stronger co-localization with CENP-C under control conditions than B56g (compare Figure 2—figure supplement 1B to Figure 2—figure supplement 1D).

The figure legend of Figure 2—figure supplement 1 (CB-Sgo1 recruits both B56a and B56g to centromeres) may be wrong. Apparently, what is meant is.….to inner Kts.

Figure 3—figure supplement 2: There is hardly any signal for wt B56g (Figure 3—figure supplement 2A) that colocalizes with CENP-C. Could the authors please comment on this?

One of the reviewers raised a concern that the data are presented in a very compact manner that may not be readily accessible to a broad readership. While no specific suggestion was provided, the authors should consider this point and consider changes that will improve readability and raise the paper's general interest.

A point emerged strongly in the post-review discussion, namely that quantitative evidence corroborating claims on the role of the newly identified sequence motifs would not simply be nice to have but would strongly reinforce the paper. An analysis in vitro with purified B56a and B56g and peptides containing the relevant sequence motifs should aim to assess whether Sgo2 reduces the binding of B56a to BubR1, whether Sgo1 binds directly to Bubr1-B56, and whether these interactions are affected by mutation of the EPVA or TKHG motifs. Based on a significant body of previous work, we believe that these experiments are feasible and can be carried out in a time compatible with an eLife revision.

We thank the reviewers and editors for their very careful review and valuable comments/suggestions. In general, we agreed that all of the proposed changes would add value to the manuscript. However, as discussed in a subsequent email exchange with the editor, we also believe that the suggestion to use purified components could not be achieved in a timeframe that is compatible with an eLife review. This decision was reached for the following reasons:

The proposed work would require the purification of 7 different components (BubR1, B56α, B56γ, PPP2CA, PPP2R1A, Sgo1 and Sgo2) followed by the phosphorylation of one of these components (BubR1) with two kinases (Plk1/Cdk1) in an attempt to generate two stable complexes: (1) Sgo1:pBubR1:PP2A-B56γ, and (2) Sgo2:PP2A-B56α. This second complex could then be tested for its ability to bind to phospho-BubR1. These in vitro assemblies would also then need to be carried out in the presence of peptides against our identified motif to see if complex assembly was prevented. In addition to the obvious time constraints, we were concerned that assembling phospho-dependent interactions in the presence of a purified phosphatase may be difficult, and we would also have no indication as to whether the peptides could adopt a conformation that binds to Sgo1 or Sgo2 with sufficient strength to prevent the key interactions.

Nevertheless, although these in vitro experiments did not seem feasible to us, we did still clearly see the value of trying to address the main questions set out in the review. Therefore, as discussed in our previous email exchange, we have attempted to address these key questions (paraphrased below for clarity) by alternative means:

1) Does Sgo2 inhibit the ability of B56α to bind to BubR1?

2) Does Sgo1 bind directly to the BubR1-B56 complex?

3) Are any effects identified in 1 or 2 dependent on the EPVA/TKHG motif?

4) Can centromeric cohesion be maintained by only B56α, and not B56γ, as our localisation data would predict.

Below is a summary of the new data that is now included in the manuscript to address these key questions:

1) Does Sgo2 inhibit the ability of B56a to bind to BubR1?

We have now examined the effect of Sgo2 depletion on the ability of B56α to bind to BubR1 and other LxxIxE-containing proteins. This new data (now included as Figure 6—figure supplement 2) shows that efficient Sgo2 depletion does not enhance the ability of B56α to bind to either BubR1 or other proteins with an LxxIxE motif. Furthermore, we are unable to detect clear Sgo2 signal in any B56 IPs, even though LxxIxE interactions are clearly different between B56α and B56γ. We now have four independent pieces of data to suggest that Sgo2 does not inhibit B56α binding to BubR1. We copy a paragraph from the new Discussion section that expands on this point:

“…a simple explanation could be that Sgo2 interaction obscures the LxxIxE binding pocket. However, this appears unlikely for four reasons: (1) Sgo2 depletion does not relocalise B56α to kinetochores (Figure 2A,B), (2) Sgo2 depletion does not enhance the ability of B56α to bind to BubR1 or other LxxIxE motifs during mitosis (Figure 6—figure supplement 2), (3) centromere and kinetochore binding can occur together in certain B56α-γ chimaeras (Figure 6—figure supplement 1B), and (4) the regions that define each of these localisations do not fully overlap (Figure 6G). Although we believe these results suggest that Sgo2 is unlikely to block LxxIxE interaction, in vitro experiments with purified components would ultimately be needed to formally rule this out.”

2) Does Sgo1 bind directly to the BubR1-B56 complex?

Current structural data suggests that Sgo1 and B56 could interact simultaneously with BubR1, and our data indicates that a BubR1-B56-Sgo1 complex could potentially exist at kinetochores. Considering that Sgo1 binds directly to the PP2A-B56 complex, we reasoned that we could address this question by immunoprecipitating BubR1-wt or -dKARD (which cannot bind B56). If Sgo1 is detected in BubR1-wt, but not BubR1-dKARD IPs, then this would suggest that BubR1 requires B56 for Sgo1 interaction; thus, implying the existence of a BubR1-PP2A-B56:Sgo1 complex.

We therefore immunoprecipitated BubR1-wt or dKARD from nocodazole-arrested cells and probed for Sgo1 and B56. The figure below shows that we could detect B56γ, but not B56α, in immunoprecipates from only BubR1-wt cells, as predicted. Unfortunately, however, we were unable to detect Sgo1 in any of these IPs, even after scaling up the input and performing rapid purifications (with a GFP-nanobody) to try to detect transient interactions. Therefore, we cannot currently state whether Sgo1 does not form a complex with BubR1-B56 in cells, or whether this complex is simply not stable enough to be detected following immunoprecipitation. We suggest to keep this as “results not shown” and have added the following new paragraph to the discussion:

“We were unable to detect Sgo1 in YFP-BubR1 immunoprecipitates (results not shown), however, this could simply reflect an interaction that is either transient or unstable away from kinetochores. It will be important in future to clarify exactly how Sgo1 collaborates with BubR1 to control B56 localisation and, in particular, to determine whether Sgo1 can interact with BubR1:PP2A-B56 complexes directly.”

Author response image 1

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We would like to stress that we do not see this question as critical to our study at all. It follows on from a novel observation that we made regarding kinetochore B56 recruitment, but this does not impact on isoform-specific localisation at all (Sgo1 binds all B56 isoforms equally well: see Figure 2—figure supplement 1). That is why we believe this question is suitable to be left as a future direction.

3) Are any effects identified in 1 or 2 dependent on the EPVA/TKHG motif?

As outlined in point 1 above, Sgo2 is very unlikely to affect LxxIxE/BubR1 binding. Therefore, although the EPVA motif is definitely required for Sgo2 binding (Figure 6A-D), this is unlikely to impact on LxxIxE binding. In addition, we believe that the EPVA/TKHG motif cannot strongly influence Sgo1 binding because Sgo1 can bind to both B56α and B56g (Figure 2—figure supplement 1).

4) Can centromeric cohesion be maintained by only B56α, and not B56γ, as our localisation data predicts.

We were actually very surprised that this was not considered an essential revision, given than it is not possible to formally demonstrate a true division of labour between B56 isoforms – as our title states – without this information. Furthermore, in our opinion, this was perhaps the most novel aspect of our study given that there are no previous demonstration that B56 isoforms can control separate cellular processes. This particular advance was not commented on in the summary by the reviewers, which we suspect is the main reason that this point was not seen as essential.

Nevertheless, we now include this important new data in Figure 1c, which demonstrates that, as predicted previously, depletion of all B56 isoforms leads to premature sister chromatid splitting, and this can be fully rescued if B56α is retained, but not if only B56γ remains.

Other important points:

For some intensity plots, the authors indicate if the observed differences are significant or not (Figure 2, Figure 3B and D, Figure 2—figure supplement 1, Figure 3—figure supplement 2, Figure 6) but for others they do not (Figure 3H and J, Figure 3—figure supplement 1). Please add this information to all the plots.

Thank you for pointing out this omission. The correct stats have now been added to all the Figures.

In some of the presented images, a clear-cut centromeric or kinetochore localization of B5a and B56g, respectively, as compared to the marker proteins Sgo2 and CENP-C, was not immediately recognisable (see for example Figure 1B). This is particularly true for endogenously tagged B56g, which is almost equally abundant at kinetochores and centromeres (see Figure 1—figure supplement 3D). At the very least, the partially overlapping localisation should be acknowledged.

We agree that it is not possible to stringently categorise localisation based on the results presented. The text has now been modified to make careful statements such as predominantly or mainly centromeric/kinetochore.

Kinetochore associated B56g, but not centromeric B56a, was sufficient to maintain the kinetochore functions of PP2A-B56 after the knockdown of all other B56 isoforms. The authors should explicitly state here that this cannot be explained by a lower expression level of B56a in these cells, as is only shown later in Figure 4A (but never commented upon).

A statement has been added to the text to explain that B56α and B56γ are both consistently expressed in HeLa cells (in relation to Figure 1—figure supplement 2 and Figure 1—figure supplement 3). Furthermore, the new data indicates that preserving B56α can only retain centromeric functions, whereas retaining B56γ only preserves kinetochore functions. This removes any concerns that the observed effects might be due to low level expression of one particular isoform.

The reverse experiment, addressing whether B56a alone, but not B56g, can maintain the centromeric functions of PP2A-B56 is lacking and the authors could elect to carry it out.

Indeed, we agree that this was a very important omission given that the title of the manuscript refers to shared labours between B56 isoforms at the kinetochore and centromere. New data has now been included to demonstrate that B56γ cannot support centromeric cohesion, whereas B56a can (Figure 1C).

The relative kinetochore intensity (e.g. Figure 1—figure supplement 5C) apparently refers to the combined kinetochore + centromere targeting. The normalised kinetochore intensity then shows the distribution between kinetochores and centromeres (e.g. Figure 1—figure supplement 5D). The y-axes should be re-defined to make this difference clear.

The axes and graph titles have now been changed to clarify this point.

Figure 1—figure supplement 2. Please include the WB for the other B56 isoforms.

A new western has now been inserted to include all B56 isoforms, except for B56β which was undetectable.

In Figure 2 and Figure 2—figure supplement 1, the authors aim to demonstrate that specific binding of B56a to Sgo2 contributes to its centromeric localization. With these figures, the following problems were identified:

Figure 2C: Upon Sgo1 depletion, B56a seems to change its localization from a primarily centromeric localization to a primarily kt localization. Yet, this is not reflected in the quantifications shown in 2D and 2E.

The graph in Figure 2D shows relative intensity of centromere and kinetochore staining combined, which is why this does not change following Sgo1 depletion (B56a simply redistributes from centromere to kinetochore under this condition). The previous axis indicated “kinetochore intensities”, which was incorrect since the centromere/kinetochore cannot be distinguished with this type of analysis. All of these axes have therefore now been changed to read “Cen/Kt intensity” to avoid confusion.

The lineplots in Figure 2E allows the Cen/Kt can be visualised separately, and this demonstrates that B56a redistributes towards the kinetochore under this condition: the new Figure 2E now contains additional repeats to reduce variability and this does indeed demonstrate redistribution towards the kinetochore.

Furthermore, Figure 2D gives the impression that Sgo1 is very efficiently depleted in Sgo1RNAi cells. The IF image shown in C gives a different impression. Could the authors please comment on this?

We try to always choose images that most closely represent the mean data (a statement is included in the methods to indicates this). In the previous Figure 2D the punctate staining was actually background staining that do not consistently align with kinetochores and can also be seen in the siCon image. However, the punctate pattern visible in the zoom was indeed confusing since it did partially overlap with kinetochore. Therefore, a different region has now been selected to avoid confusion and to better represent the minimal kinetochore staining seen under this condition.

Subsection “Sgo2 provides specificity for centromeric B56 recruitment”: "…, CB-Sgo2 was only able to recruit B56a (Figure 2h-k)." This statement is puzzling. Figure 2J shows hardly any YFP-B56g localization. Why is the localization so different compared to Figure 1 and how was the quantification based on such IF images performed?

Thank you for pointing out this error. This image was not representative of the data because it had high background cytosolic levels and poor kinetochore staining. A new example image has now been included that better represents the mean data.

Importantly, it should be clarified whether this figure and the related Figure 2—figure supplement 1 are carried out under conditions of depletion of Sgo1 and/or Sgo2, as this does not seem to be stated anywhere.

The CB-Sgo1/2 experiments were all performed in the presence of endogenous Sgo1/2. This has now been clarified in the Materials and methods section.

Subsection “Sgo2 provides specificity for centromeric B56 recruitment”: "…CB-Sgo1 was able to localize both B56a and B56g to the inner kinetochore (Figure 2—figure supplement 1)." This statement is not supported by the data. Under wt conditions, B56g already colocalizes with CENP-C as shown in Figure 1A and 1B. Why are the quantifications (Figure 2K and Figure 2—figure supplement 1D) so different for control conditions?

And given that B56g colocalizes with CENP-C under wt conditions, how do the authors come to the conclusion that CB-Sgo1 recruits B56g to inner kts?

Regarding points 3 and 4:

Under wt conditions, B56a and B56g already colocalise with the centromere or kinetochore, respectively (Figure 1A and B). The later figures with CB-Sgo1/2 (Figures2k, S6d etc.) still contain this baseline Cen/Kt localisation, but we then quantify the additional localisation induced by CB-Sgo1/2 expression. The same strategy was previously used by Meppelink et al., 2015 to show recruitment of B56d to CB-Sgo1. Our data indicate that CB-Sgo2 increases B56a (Figure 2I), but not B56g (Figure 2K), whereas CB-Sgo1 causes a large increase in both B56a and B56g at the Cen/Kt (Figure 2—figure supplement 1). Therefore, although the background Cen/Kt does complicate the overall interpretation (this is now clarified in the text), we still believe that our previous statements were supported by the data; given that we observe statistically significant increases only in certain conditions (i.e. in all CB-Sgo1 or -2 conditions, except for CB-Sgo2 + B56g). Please note, that we chose to use the inner kinetochore for these experiments in spite of these difficulties, because it may be important given that Aurora B activity was previously reported to be required for Sgo2:B56 interaction (Tanno et al., 2010).

Along the same line, B56a shows a stronger co-localization with CENP-C under control conditions than B56g (compare Figure 2—figure supplement 1B to Figure 2—figure supplement 1D).

Thank you for pointing this out. The image in S6D did not demonstrate the clear kt localisation in B56g controls. This image has now been changed for a more representative alternative (now Figure 2—figure supplement 1B and D).

The figure legend of Figure 2—figure supplement 1 (CB-Sgo1 recruits both B56a and B56g to centromeres) may be wrong. Apparently, what is meant is.….to inner Kts.

As stated above, we have now correctly labelled all these axes to indicate “Cen/KT intensities” since we cannot distinguish between these two localisations in this type of analysis.

Figure 3—figure supplement 2: There is hardly any signal for wt B56g (Figure 3—figure supplement 2A) that colocalizes with CENP-C. Could the authors please comment on this?

The cells imaged in Figure 3—figure supplement 2A are in interphase. Although B56γ is present in the nucleus in interphase, its main kinetochore receptor, BubR1, is not.: BUBR1 is only recruited to the kinetochore following nuclear envelope breakdown (see Nijenhuis et al., 2014). As such, in the wt situation prior to NEB, B56γ does not colocalize with CENP-C at kinetochores.

One of the reviewers raised a concern that the data are presented in a very compact manner that may not be readily accessible to a broad readership. While no specific suggestion was provided, the authors should consider this point and consider changes that will improve readability and raise the paper's general interest.

We thank the reviewer for this comment. We have now expanded the text slightly in key areas to explain things better to readers who do not have experience in kinetochore signalling. Hopefully this will now help to raise interest from a general audience.

Author details

1. Giulia Vallardi

   Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

2. Lindsey A Allan

   Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Lisa Crozier

   Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, United Kingdom

   Contribution

   Methodology

   Competing interests

   No competing interests declared

4. Adrian T Saurin

   Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, United Kingdom

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   a.saurin@dundee.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9317-2255

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Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.