PP2A-B56 regulates Mid1 protein levels for proper cytokinesis in fission yeast

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Abstract

Protein phosphorylation regulates many steps in the cell division process including cytokinesis. In the fission yeast S. pombe , the anillin-like protein Mid1 sets the cell division plane and is regulated by phosphorylation. Multiple protein kinases act on Mid1, but no protein phosphatases have been shown to regulate Mid1. Here, we discovered that the conserved protein phosphatase PP2A-B56 is required for proper cytokinesis by promoting Mid1 protein levels. We find that par1 Δ cells lacking the primary B56 subunit divide asymmetrically due to the assembly of misplaced cytokinetic rings that slide toward cell tips. These par1 Δ mutants have reduced whole-cell levels of Mid1 protein, leading to reduced Mid1 at the cytokinetic ring. Restoring proper Mid1 expression suppresses par1 Δ cytokinesis defects. This work identifies a new PP2A-B56 pathway regulating cytokinesis through Mid1, with implications for control of cytokinesis in other organisms.

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    Referee #3

    Evidence, reproducibility and clarity

    Protein kinases play an important role in regulating cell division and the dynamics of the actomyosin ring both in yeast and mammalian cells. While this raises the possibility that regulated protein dephosphorylation may also affects the division dynamics, few experimental studies have been reported. In the current study, Chrupcala and Moseley screen 9 non-essential protein phosphatases and show that the conserved protein phosphatase PP2A-B56 regulates division plane positioning in fission yeast. The authors show that Par1 localizes to the division site and that cells lacking Par1 have impaired levels of regulators of actomyosin ring positioning, including the anillin-like protein Mid1. Interestingly, restoring wildtype Mid1 levels partially suppresses the division defects observed in the par1∆ mutant.

    The study is well-executed with attention to detail and careful phenotype quantification and analyses. The conclusions of the study are largely supported by experiments to yield the novel insights. This reviewer commends the authors on reporting the negative results regarding Mid1-3xSLiM mutant (Results section) and failure to detect physical interaction between Par1 and Mid1 (Discussion section). The authors provide an interesting discussion. Furthermore, they describe very relevant additional experiments but they restrict their execution to future studies that complement the current manuscript

    Major concern

    The authors show that par1∆ cells have impaired levels of Mid1, Cdc15 and Rga7, yet propose that Mid1 reduction is the principal cause of cytokinetic defects in par1∆ cells. Even though increase in Mid1 levels rescues par1∆ defects, it is possible that increased Mid1 levels suppress another primary defect (e.g. Rga7 increased levels). Thus, it would be interesting to perform the following two experiments:

    1. The authors in the discussion say "we found that cytokinesis defects arise from decreased Mid1 levels" but this is not formally shown other than in par1∆ cells. Thus I would suggest monitor cytokinesis in cells with Mid1 levels directly reduced to levels comparable to those observed in par1∆ cells (as quantified in Fig 3B). Monitoring the effects of reduced Mid1 levels on Rga7 and Cdc15 would also be interesting.
    2. Monitoring the levels of Rga7 and Cdc15 in par1∆ cells rescued by the second copy Mid1.

    Minor concern

    Even though definitive evidence for Par1 mechanism of Mid1 regulation might be difficult to obtain, the authors may choose to strengthen their work on the role of dephosphorylation at the actomyosin ring. For example, using the GFP-GFPnanobody pairing to force interaction between Mid1 and Par2 in par1∆ cells may provide support for dephosphorylation playing a more direct role in actomyosin ring positioning.

    Referees cross-commenting

    Consultation regarding Review 1:

    Reviewer 3 shares the interest in points 1,3,5.

    Relating to point 2: Not sure what the reviewer specifically wishes here.

    Relating to point 4: Extensive analyses of many cytokinetic proteins in par1∆ cells, and importance of their levels for cytokinesis, would be interesting but perhaps beyond the scope of this study. I believe it would suffice to monitor Cdc15, Bgs1 and Rga7 in the 2xMid1 rescue that authors performed.

    Consultation regarding Review 2: Reviewer 3 shares the interest in major points 1 (though authors do leave open the possibility that PP2A acts indirectly),3,4,5. Regarding point 2: This might be difficult to ascertain directly and instead it might suffice to show that Mid1 levels reduced to those observed in par1∆ phenocopy the par1 mutant's division defects.

    Significance

    The study is well-executed with novel findings on regulation of the cell division and the physiological roles of phosphatases. The study will benefit cell polarity and cell division research fields as well as researchers interested in roles of protein phosphatases.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this study, the authors investigates the functional relationship of Mid1, which plays a central role in the positioning of the contractile ring (CAR) in fission yeast, with protein phosphatases. Previous studies have shown that Mid1 is phosphorylated and modified by several kinases, but its relationship with phosphatases is not well understood except of Clp1. Therefore, the authors examined whether phosphorylase gene disruption strains were able to divide symmetrically. The authors found that cells lacking par1, which encodes the regulatory subunit of PP2A, frequently divided asymmetrically. Furthermore, they found that the par1 deletion mutant showed reduced protein levels of Mid1 in the cells. Furthermore, they showed that increasing the expression level of Mid1 can partially compensate for the mitotic abnormalities of the par1 strain. Since Par1 localizes to the division site, the authors also investigated the possibility of binding with Mid1, focusing on its SLiM motif. However, at this time, it is unclear whether direct binding of Par1 to Mid1 is necessary for Mid1 to exert its cellular functions, since no conspicuous cytokinesis abnormalities were observed in Mid1 with the mutation in the SLiM motif.

    The focus of the study to be interesting and the clarity of the overall argument of the manuscript to be almost adequate. However, the authors should investigate or mention the following points. Also, a more appropriate and convincing way of presenting experimental results is required.

    Major comments:

    1. Does Par1 (PP2A) work in the dephosphorylation of Mid1? Looking at the band pattern of the western blot of Mid1 in Fig. 3E, there are several bands. This band shift probably indicates phosphorylation of Mid1, but is there any difference in the band shift between the wild-type strain and the par1 gene disrupted strain? Mid1 is phosphorylated in a cell cycle-dependent manner. So, it would be interesting to examine the cell cycle-dependent phosphorylation pattern using synchronous culture.
    2. In Fig. 5A, par1- and ppa2-deficient strains show little nuclear localization of Mid1 in interphase cells. If little or no Mid1 enters the nucleus, could it be possible that the mitotic abnormality is not due to a reduction in Mid1 protein levels, but is directly due to an effect on the shuttling of Mid1 between the cell nucleus and cell surface?
    3. The photographs showing cellular localization of proteins, such as Figs. 3, 4, 5, 6B, and 6C, are not convincing. The authors should show a typical picture of the cell population as supplemental figures, rather than trimming a single cell.
    4. Is the Mid1-deficient strains impaired in the localization of Par1 to the division plane? As shown in Fig. 3A, the fluorescence of Cdc15 and Rga7 is predominantly enhanced in the par1 deletion strain (Fig. S2 also shows a protein with a significant difference in localization). The authors should consider these points and make more comprehensive discussion.
    5. Fig. 3D and part of Fig. 5B also use the same photograph. This raises the suspicion that these data were taken only once. If so, the authors should do replicated experiments to assure the authenticity of the data. In addition, perhaps it is a careless mistake, but this way of presenting data should not be allowed unless specifically mentioned in the manuscript.

    Minor point.

    Typos. p. 11 Mid1-13my levels,.

    Lack of uniformity in description p. 13 Methyl-2-benzimidazole and p. 15 methyl-2-benzimidazole In the figure of the paper Mid1-mNeonGreen and mid1-mNeonGreen

    Significance

    The importance of this study is that it deepens our knowledge of Mid1 protein of fission yeast, an important model organism for the study of cytokinesis. Although the possibility that Par1 is involved in the maintenance of Mid1 protein levels has been demonstrated, the molecular mechanism has not been clarified. It might be expected that anilline, which is similar to Mid1 in animal cells, might have a similar relationship to protein phosphatases, but there is no evidence for this. Therefore, at this moment, the effect of these findings may be limited to the fission yeast research community.

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    Referee #1

    Evidence, reproducibility and clarity

    The study by Chrupcala and Moseley explores the role of the protein phosphatase PP2A-B56 in cytokinesis, particularly focusing on its regulatory subunit Par1 and its interaction with Mid1 in fission yeast (S. pombe). The authors report that in cells lacking Par1 (par1d), the contractile rings are mispositioned, and the levels of multiple cytokinetic proteins, including Mid1, are altered, leading to defects in cytokinesis. They suggest that the reduction in Mid1 levels is responsible for the observed cytokinetic defects and show that restoring Mid1 levels can partially rescue these defects. The study further demonstrates that Mid1 shuttling between the nucleus and cell membrane is impaired in par1d mutants. Thus, par1d seems to cause a range of defects, from altered protein levels to specifically impairing Mid1 functions. However, the study does not demonstrate a direct interaction between Par1 and Mid1, leaving the exact mechanism by which Par1 regulates Mid1 levels unclear.

    The manuscript is well-written and investigates the lesser-studied role of protein phosphatases in cytokinesis, which is of general interest. However, a major criticism is the lack of evidence supporting a direct role of PP2A-B56 and its regulatory subunit Par1 in cytokinesis. It is possible that the phenotype observed in par1d cells results from a global impairment of protein regulation rather than a specific effect on Mid1. Indirect factors, such as changes in cell size/shape, could also produce similar phenotypes.

    To strengthen the manuscript, the authors should consider the following points:

    1. Changes in cell size/shape can influence Mid1 levels and nucleus positioning. Many par1d cells shown in the manuscript have bulgy morphologies. The manuscript should address whether cell size/morphology differences contribute to the observed phenotypes.
    2. The quantification of nucleus positioning needs further consideration, as the positioning of the nucleus has been shown to be a function of cell size, etc.
    3. The manuscript proposes that Par1 acts by reducing Mid1 levels and that restoring Mid1 levels can rescue the defects. However, given the known mechanisms of Mid1 localization, it is unclear why a reduction in Mid1 levels alone would lead to cytokinetic defects. To establish that the defects are due to Mid1 reduction, the authors should demonstrate that lowering Mid1 levels (e.g., using a low-expression promoter) phenocopies the cytokinesis defects observed in par1d cells.
    4. As shown in par1d cells, other cytokinesis proteins have impaired levels. It should be investigated whether changes in the levels of other proteins (e.g., Cdc15, Rga7, Myp2, etc.) in par1d cells contribute to the cytokinetic defects. In general, it is not discussed how the par1d mutant can lead to differential levels of many cytokinesis proteins. Does the reduction of Mid1 explain this shift? It is conceivable that changes in the levels of other proteins could also produce similar cytokinesis defects.
    5. The manuscript would benefit from additional functional rescue experiments. For example, expressing a phosphatase-dead version of Par1 in par1d cells could help determine if its phosphatase activity is necessary for cytokinesis.

    Significance

    The study by Chrupcala and Moseley explores the role of the protein phosphatase PP2A-B56 in cytokinesis, particularly focusing on its regulatory subunit Par1 and its interaction with Mid1 in fission yeast (S. pombe). The authors report that in cells lacking Par1 (par1d), the contractile rings are mispositioned, and the levels of multiple cytokinetic proteins, including Mid1, are altered, leading to defects in cytokinesis. They suggest that the reduction in Mid1 levels is responsible for the observed cytokinetic defects and show that restoring Mid1 levels can partially rescue these defects. The study further demonstrates that Mid1 shuttling between the nucleus and cell membrane is impaired in par1d mutants. Thus, par1d seems to cause a range of defects, from altered protein levels to specifically impairing Mid1 functions. However, the study does not demonstrate a direct interaction between Par1 and Mid1, leaving the exact mechanism by which Par1 regulates Mid1 levels unclear.

    The manuscript is well-written and investigates the lesser-studied role of protein phosphatases in cytokinesis, which is of general interest. However, a major criticism is the lack of evidence supporting a direct role of PP2A-B56 and its regulatory subunit Par1 in cytokinesis. It is possible that the phenotype observed in par1d cells results from a global impairment of protein regulation rather than a specific effect on Mid1. Indirect factors, such as changes in cell size/shape, could also produce similar phenotypes.