SDS-22 stabilizes the PP1 catalytic subunits GSP-1/-2 contributing to polarity establishment in C. elegans embryos

  1. Yi Li
  2. Ida Calvi
  3. Monica Gotta

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

In many cells, cell polarity depends on the asymmetric distribution of the conserved PAR proteins, maintained by a balanced activity between kinases and phosphatases. The C. elegans one-cell embryo is polarized along the anterior-posterior axis, with the atypical protein kinase C PKC-3 enriched in the anterior, and the ring finger protein PAR-2 in the posterior. PAR-2 localization is regulated by PKC-3 and the PP1 phosphatases GSP-1/-2. Here, we find that, like GSP-2 depletion, depletion of the conserved PP1 interactor SDS-22 results in the rescue of the polarity defects of a pkc-3 temperature-sensitive mutant. Consistent with the rescue, SDS-22 depletion or mutation results in reduced GSP - 1/ - 2 protein levels and activity. The decreased levels of GSP-1/-2 can be rescued by reducing proteasomal activity. Our data suggest that SDS-22 regulates polarity not by directly regulating the localization or activity of GSP-1/-2, but by protecting these PP1 catalytic subunits from proteasome-mediated degradation, supporting recent data in human cells showing the SDS22 is required to stabilize nascent PP1.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Manuscript number: RC- 2025-02880

    Corresponding author(s): Monica, Gotta

    1. General Statements [optional]

    We thank the reviewers for their useful comments that will improve our manuscript and make it clearer. We agree with Reviewer 1 that SDS-22 has more general functions in cellular processes by maintaining GSP-1/-2 levels, rather than only regulating cell polarity. We have now modified our conclusion in the text (all changes are highlighted in yellow) and we hope that it is now more clear and better explained. Below we address the reviewer’s comments one by one and indicate how we have or will address the comments in the final version. We expect the revisions to take 2-3 months.

    2. Description of the planned revisions

    Major comments

    Reviewer 1

    (1) Overall, the evidence supporting the core finding that SDS-22 is required for normal GSP-1/2 levels is strong and well documented. The experiments were performed well and controls, statistics, replicates were appropriate. Our only slight reservation was whether the effect of sds-22(RNAi) on stability may be overstated due to the use of GFP fusions to GSP-1/2 for this analysis. The authors note these alleles are hypomorphic, potentially raising the possibility that GFP tags destabilise the proteins and make them more prone to degradation. Ideally this would be repeated with an untagged allele via Western (e.g. Peel et al 2017 for relevant antibodies).

    We thank the reviewer for the general comments. To address this important point on the protein levels we have requested GSP-1 and GSP-2 antibodies reported in Peel et al and Tzur et al (Peel* et al, 2017; Tzur et al*, 2012). The published GSP-1 antibody has been used in western blot, and the GSP-2 antibody has been used in both immunostaining and Western blot analysis. Despite our efforts, we were not able to detect GSP-2 neither on western blots nor on immunostainings with the aliquot we have received. On the opposite, GSP-1 antibodies worked well on western blot. We have already measured the GSP-1 levels in SDS-22 depleted embryos (N=2, see below) and we observed reduced levels, confirming our initial result. However, as the reviewer rightly pointed out, the levels are reduced by 20% (rather than about 50% as in the GFP strain), suggesting that indeed the GFP fusion does contribute to the instability. We will measure GSP-1 levels in at least an additional sds-22(RNAi) experiment and in sds-22(E153A) embryos.

    Left, Western Blot of embryonic extracts from *N2 *in *ctrl(RNAi) *and sds-22(RNAi) embryos. Tubulin is used as a loading control. Right, Fold change of GSP-1 normalized to Tubulin levels. N = 2.

    Since we could not detect endogenous GSP-2 with the antibodies we have received, we will generate an OLLAS-tagged GSP-2 strain. OLLAS is a commonly used tag consisting of 14 amino acids (Park* et al*, 2008), with an additional 4 amino acids as a linker. The tag is much smaller than mNeonGreen, which consists of approximately 270 amino acids. We will then measure the GSP-2 levels using the ollas antibody in sds-22(RNAi) embryos. We will also cross this strain with *sds-22(E153A) *and measure OLLAS::GSP-2 levels in this mutant. If this strain is not embryonic lethal, as in the case of the *mNG::gsp-2; sds-22(E153A) *(Fig EV6A), it will also suggest that ollas::gsp-2 does not behave as hypomorph.

    These data will complement the data shown in Fig 6.

    (2) The role for SDS-22 in polarity is rather weak. Both the SDS-22 depletion phenotypes and the ability of SDS-22 depletion to suppress pkc-3(ts) polarity phenotypes are modest (and weaker in than GSP-2 depletion). For example, the images in Figure 1B appear striking, but from Movie S1 it is clear that this isn't a full rescue as PAR-2 is initially uniformly enriched on the cortex (rather than mostly cytoplasmic) and it is never fully cleared. In the movie, the clearance at the point of pronuclear meeting is very modest. Quantitation might be helpful here (i.e. as in Figure 3G). As the authors state, it seems that SDS-22 does not have a specific role in polarity beyond the general effect on GSP-1/2 levels. This does not undermine the core message of the paper, but we would recommend downplaying the conclusions with respect to contributing to polarity establishment. For example "...suggesting that SDS-22 regulates GSP-1/-2 activity to control the loading of PAR-2 to the posterior cortex in one-cell stage C. elegans embryos" implies a regulatory role for SDS-22 in polarity, but we would interpret it as simply helping reduce aberrant degradation of GSP-1/2 and this impacts a variety of cellular processes including polarity.

    We agree with the reviewer that the rescue of pkc-3ts polarity defects by SDS-22 depletion is not as strong as GSP-2 depletion, and as suggested, we have re-quantified the phenotype, as we did in Fig 3G, as shown below in Fig 1C.

    This has replaced Fig.1 in the manuscript.

    Accordingly, we have clarified this in the text in several locations. We have added “partial” rescue in many places and modified conclusions in the results and discussion. The changes are all highlighted and the major ones are also below:

    From Result Line 119-121, page 5:

    “In contrast, depletion of SDS-22 resulted in PAR-2 localization being restricted to the posterior cortex in 87.5% of the one-cell stage embryos (Fig 1B) and PAR-2 was localized to the P1 blastomere after the first cell-division (Movie EV1).”

    To: Result Line 122-125, page 5

    “In contrast, depletion of SDS-22 resulted in PAR-2 localization being enriched in the posterior cortex in 87.5% of the one-cell stage embryos (Fig 1B,C) and PAR-2 was localized to the P1 blastomere after the first cell-division (Movie EV1).”

    From Result Line 172-175, page 7:

    “Our data show that depletion of SDS-22 results in a smaller PAR-2 domain, suppresses the polarity defects of a *pkc-3 *temperature sensitive strain and the aberrant PAR-2 localization observed in the PAR-2(L165V) mutant strain. As SDS-22 is a conserved PP1 regulator, our data suggest that SDS-22 positively regulates GSP-2 in polarity establishment.”

    To: Result Line 178-181, page 7

    “Our data show that depletion of SDS-22 results in a smaller PAR-2 domain, partially suppresses the polarity defects of a pkc-3 temperature sensitive strain and the aberrant PAR-2 localization observed in the PAR-2(L165V) mutant strain. As SDS-22 is a conserved PP1 regulator, our data suggest that SDS-22 positively regulates GSP-2.”

    From Result Line 256-257, page 10:

    “suggesting that the interaction of SDS-22 with the PP1 phosphatases is important for polarity establishment.”

    To: Result Line 264-265, page 10

    “suggesting that the interaction of SDS-22 with the PP1 phosphatases contributes to polarity establishment”

    From Result Line 311-313, page 12:

    To conclude, while our genetic data on PAR-2 cortical localization suggest that SDS-22 is not required to fully activate GSP-1 and/or GSP-2, depletion or mutation of SDS-22 results in a reduced activity of the phosphatases.

    To: Result Line 319-322, page 12

    To conclude, while our genetic data on PAR-2 cortical localization suggest that SDS-22 is not required to fully activate GSP-1 and/or GSP-2, depletion or mutation of SDS-22 results in a reduced activity of the phosphatases, as shown by phospho-histone H3 (Ser10) levels. This suggests that SDS-22 plays a general role in regulating GSP-1 and GSP-2, which is not specific to cell polarity.

    From Result Line 391-392, page 15:

    In summary, our results show that SDS-22 maintains the levels of GSP-1 and GSP-2 by protecting them

    392 from proteasome mediated degradation.

    To: Result Line 402-403, page 15

    In summary, these data show that SDS-22 is important to maintain the levels of GSP-1 and GSP-2 by protecting them from proteasome mediated degradation.

    We have also rephrased our conclusion according to Reviewer 1’s suggestion.

    From Introduction Line 95-101, Page 4:

    Here we show that SDS-22 depletion rescues the polarity defects caused by reduced PAR-2 phosphorylation in the pkc-3(ne4246) mutant at the semi-restrictive temperature (24°C), similarly to the depletion of GSP-2. Depletion of SDS-22 results in lower GSP-1 and GSP-2 protein levels which can be rescued by depleting proteasomal subunits. These results establish SDS-22 as a regulator of PAR polarity establishment in the C. elegans one-cell embryo and are consistent with and complement the recent data in mammalian cells showing that SDS22 is important to control the stability of the PP1 phosphatase (Cao* et al.*, 2024).

    To: Introduction Line 96-101, Page 4

    *Here we show that SDS-22 depletion partially rescues the polarity defects caused by reduced PAR-2 phosphorylation in the pkc-3(ne4246) mutant at the semi-restrictive temperature (24°C). Depletion of SDS-22 results in lower GSP-1 and GSP-2 protein levels which can be rescued by depleting proteasomal subunits. These results establish that SDS-22 contributes to cell polarity by regulating GSP-1/-2 levels and are consistent with and complement the recent data in mammalian cells showing that SDS22 is important to control the stability of the PP1 phosphatase (Cao et al., 2024). *

    From Discussion Line 417-420, page 17:

    Depletion of SDS-22, or mutation of its E153 residue (E153A) important for SDS-22-PP1 interaction resulted in reduced GSP-1/-2 protein levels, decreased dephosphorylation of a PP1 substrate, and a smaller PAR-2 domain, suggesting that SDS-22 regulates GSP-1/-2 activity to control the loading of PAR-2 to the posterior cortex in one-cell stage* C. elegans* embryos.

    To: Discussion Line 426-429, page 17

    *Here we find that a conserved PP1 regulator, SDS-22, when depleted, results in a smaller PAR-2 domain and can partially rescue the polarity defects of a pkc-3(ne4246) mutant. We demonstrate that SDS-22 contributes to the activity of GSP-1/-2 by protecting them from proteasomal degradation and maintaining their protein levels. *

    Add new discussion to Discussion Line 429-432, page 17:

    Taken together, our data suggest that the role of SDS-22 in polarity is indirect via the regulation of GSP-1/-2 levels. In support of this, SDS-22 depletion results in broader GSP-1/-2 dependent phenotypes such as increased Phospho-H3 (Ser10) (Fig 5) and centriole duplication defects in later-stage embryos (Peel et al., 2017).

    (3) Specificity of SDS-22 effects on polarity. SDS-22 (or GSP-1/2) depletion is likely to have effects on many pathways. We wondered whether some of the polarity phenotypes may not be specifically due to changes in the PAR-2 phosphorylation cycle as implied.

    One candidate is the actomyosin cortex. It was noticeable that control and sds-22 embryos were different: In Movies S1, S2, and S3 control embryos show either stronger or more persistent cortical ruffling or pseudocleavage furrows. This is also visible in Figure 3A. Is it possible that disruption of SDS-22 reduces cortical flows (time, intensity or duration) and could this explain the small reduction in anterior PAR-2 spreading and thus the slightly smaller domain size measured in Figures 1B and 3A.

    We have noticed that SDS-22 depletion results in less ruffling and reduced pseudocleavage furrows. To properly address this question we should have a condition in which we can rescue the cortical flow reduction in the SDS-22 depletion and measure the PAR-2 domain. Since we do not know how SDS-22 reduces the flows, we could not come up with a clean experiment to address this issue and are happy to have suggestions.

    We believe that the most rigorous way to address this issue, as reviewer 1 points out, is to clearly address this limitation in the text. We have now added this in the discussion:

    Discussion Line 463-466, page 18:

    Consistent with GSP-2 reduced levels, SDS-22 depleted or E153A mutant embryos also have a smaller PAR-2 domain. However, since these embryos also show reduced cortical ruffling (Movie EV1,2) and are smaller (Fig EV2C) we cannot exclude that these two phenotypes also contribute to the smaller size of the PAR-2 domain.

    A potentially related issue could be embryo size. sds-22 embryos generally seem to be smaller than wild-type (e.g. Figure 1B(left), 4A(left column), and particularly EV3). Is this consistently true? Could cell size effects change the ability of embryos to clear anterior PAR-2 domains as described in EV3? Klinkert et al (2018, biorXiv) note that reducing the size of air-1(RNAi) embryos reduces the frequency of bipolar PAR-2 domains.

    Quantification of perimeter of embryos at pronuclear meeting in live zygotes. Sample size (n) is indicated in the graph, each dot represents a single embryo and mean is shown. N = 5. The P value was determined using two-tailed unpaired Student’s t test.

    We quantified the perimeter of the embryos and as seen by quantification, there is a weak but significant decrease of size in the absence of SDS-22, and in SDS-22(E153A) mutant, as shown above. We have now added the data of the RNAi in the supplementary information and mentioned it in the results.

    Results Line 129, page 5:

    SDS-22 depleted embryos also displayed a smaller size (Fig EV2C).

    Klinkert et al reported that reducing the size of air-1(RNAi) embryos by depletion of ANI-2, a homolog of the actomyosin scaffold protein anillin, reduces the frequency of bipolar PAR-2 domains (Klinkert* et al*, 2018). In the image shown in the paper on bioRxiv, the PAR-2 domain appears small but there are no quantifications and these data have been removed from the published paper.

    From published data, a smaller embryo size does not appear to correlate with smaller PAR-2 domain. Chartier et al show that depletion of ANI-2 reduces embryo size without changing the relative anterior PAR-6 domain (Chartier* et al*, 2011), thereby suggesting that the posterior PAR-2 domain should not change either. In addition, Hubatsch et al reported that small embryos depleted of ima-3 tend to have larger PAR-2 domains, whereas larger embryos depleted of C27D9.1 exhibit smaller PAR-2 domains (Hubatsch et al, 2019), which is the opposite of what we see. We do not believe that the smaller PAR-2 domain is the important message of our paper. Our main question was whether PAR-2 was cortical or not and since GSP-2 had a smaller domain, we decided to quantify the PAR-2 domain length in the different RNAi conditions and mutants. Since RNAi of C27D9.1 which makes embryos bigger, results in a small PAR-2 domain, again we do not know how to experimentally address this question, unless the reviewer has a suggestion. As for the point above, we will clearly highlight this limitation in the discussion (see our reply to the previous point, now it is in Discussion Line 463-466, page 18).

    We would stress that these comments relate to interpreting the polarity phenotypes and do not undermine the core finding that SDS-22 stabilises GSP-1/2.

    We thank the reviewer and we hope that by performing the experiments mentioned above and by changing the text, their comments are properly addressed.

    Reviewer 2

    Major comment: Consistent with the model that PP1 activity is reduced in the absence of SDS-22, the authors show that a surrogate PP1 target (phospho-histone H3) becomes hyper-phosphorylated. To strengthen the study, the authors could consider performing an OPTIONAL experiment (see below) of assaying the phosphorylation status of PAR-2 itself, as this is proposed to be the target of both PKC-3 and PP1, and represent the mechanism of PAR-2 polarization.

    We thank the reviewer for this comment and also for pointing out that there is technical difficulty in the proposed experiment.

    We have already attempted to address this point without success in Calvi et al (Calvi* et al*, 2022), using western blot analysis (see below). For this we used the GFP::PAR-2 strain and used a GFP antibody (shown below in the left panel), as none of the anti-PAR-2 antibodies (neither the ones produced by us nor the ones produced by other laboratories) were working on western blot. We observed several bands of GFP::PAR-2 but were not able to determine if these represented phosphorylated forms or to compare the ratio of phosphorylated to unphosphorylated PAR-2. We did use λ-PPase in the embryonic extracts but we did not always observe a clear difference. We show three experiments below.

    Left, Western blots of gfp::par-2 embryonic extract in the presence or absence of λ-PPase (+/- PhosSTOP) and probed with anti-GFP and anti-Tubulin antibodies. Right, Representative images of fixed embryos with indicated genotypes at one-, two- and four-cell stages. DNA (DAPI) is gay. Scale bars, 5 μm. Anterior is to the left and posterior to the right.

    One possible explanation is that the role of GSP-1/-2 in PAR-2 dephosphorylation is specific to the very early embryos. As shown in the right panel above, despite PAR-2(RAFA) remaining cytoplasmic in one- and two-cell embryos due to lack of binding to GSP-1/-2, it can localize to internal cortices in four-cell stage embryos, similarly to the control and suggesting that in later embryos other mechanisms are intervening. One limitation of our Western Blot is that it is not possible to isolate only early embryos, which are a minority in a mixed population of embryos. This may mask difference of phosphorylation status of PAR-2 in the early stages.

    For the revision, we plan to blot PAR-2 using GFP antibody in gfp::par-2 embryo lysates, with both control and sds-22(RNAi) treatment. We will also compare the GFP::PAR-2 bands between *gfp::par-2 and gfp::par-2; sds-22(E153A) *mutant samples. We are not very hopeful and our failures with gsp-1/2 RNAi (unpublished) are why we did not try with SDS-22 but it is definitely worth giving it a go and we will.

    As for Hao et al (Hao* et al*, 2006) the result was quite clear. In this paper however, the authors used a transgene strain of PAR-2. We have never tried to use a transgene (the proteins are usually overexpressed) but we can deplete SDS-22 in a PAR-2 transgene as well and see if a difference is observed.

    Reviewer 3

    Major comments: major issues affecting the conclusions

    Overall, the authors' conclusions are supported by their data. The data and methods are presented clearly, with appropriate replicates and statistics. Here I propose two experiments to strengthen the link between some of their data and their claims. These experiments could take a month or two to complete.

    Experiment 1

    It would be helpful if the authors could show that blocking the proteasome in the zygote restores GSP-1/-2 levels in the absence of SDS-22 or even better in the SDS-22(E153A) mutant. This would provide more direct evidence to support their claim that SDS-22 regulates polarity by protecting PP1 from proteasomal degradation. While they are currently conducting this experiment in the germline, they cannot assess polarity there. However, in the zygote, they would be able to examine the PAR-2 domain (polarity). To do this, the authors could permeabilise the embryos and apply a proteasome inhibitor.

    This would be a straightforward experiment if we were using culture cells. One problem with the set up is that much of the protein of the one-cell embryo is inherited from the egg and the reduction in SDS-22 depletion or mutant happens already in the germline (Fig 6-7). Even if the proteasome is inhibited in embryos, the whole division process only takes 20 minutes and we wonder whether the timing will be sufficient to inhibit the proteasome, produce more protein and rescue the phenotype. We will try, as only this will tell us.

    One alternative approach would be to apply the proteasome inhibitor to adult worms in liquid culture for several hours before dissection. This would aim to inhibit degradation in the germline, therefore allowing us to test whether GSP-1/-2 levels are restored in the embryos with SDS-22 disruption. However, proteasome inhibition in the germline impairs oogenesis (Shimada* et al*, 2006), suggesting that we might incur in the same problem (unless we succeed in timing the inhibition).

    One additional experiment that we will try is to deplete other proteasomal subunits that result in a lower level or proteasomal activity reduction. As reported by Fernando et al (Fernando* et al*, 2022), depletion of RPN-9, -10, or -12 impairs proteasomal activity, but worms remain fertile.

    Quantification of mNG::GSP-2 and GFP::GSP-1fluorescence intensity in rpn-12, rpn-9, and rpn-10(RNAi) normalized to ctrl(RNAi). Mean is shown and error bars indicate SD. Dots in graphs represent individual embryo measurements and sample size (n) is indicated inside the bars in the graph. N = 1.

    So far, our data suggest that the GSP-1/-2 levels are weakly but significantly increased in the embryos (16.8% for GSP-2 and 12.5% for GSP-1) following RPN-12 depletion (see above). We will co-deplete RPN-12 and SDS-22 to assess if the protein levels of GSP-1/-2 are rescued. We will also deplete RPN-12 in gfp::gsp-1; sds-22(E153A) strains to test if GSP-1 levels are rescued. We cannot measure GSP-2 levels in mNG::GSP-2; sds-22(E153A) because they are embryonic lethal (see details below in the reply to minor comments of Reviewer 3).

    Left, Representative midsection images of* gfp::gsp-1 *and *gfp::gsp-1;sds-22(E153A) *embryos in *ctrl(RNAi) *and rpn-12(RNAi).__ Right, __Quantification of GFP::GSP-1 intensity levels. N = 1.

    Our preliminary data showed that similar to germlines (Fig 7G-I), RPN-12 depletion in gfp::gsp-1; sds-22(E153A) rescued the reduction of GSP-1 levels in embryos (shown above). We will perform two additional experiments to quantify GSP-1 levels.

    We will also test if the smaller PAR-2 domain in sds-22(E153A) mutant is rescued by RPN-12 depletion. With these experiments, we aim to answer if proteasome inhibition rescues the reduced levels of GSP-1/-2 and thereby rescues the reduced PAR-2 domain when SDS-22 is depleted or mutated.

    Experiment 2

    The posterior localization of PAR-2 after co-RNAi of GSP-1 and SDS-22 contrasts with the absence of PAR-2 at the cortex when both GSP-1 and GSP-2 are depleted. This difference may be due to the partial reduction of GSP-2 levels when SDS-22 is depleted, compared to the more substantial reduction of GSP-2 upon GSP-2 RNAi. Have the authors considered combining full depletion of GSP-1 with partial depletion of GSP-2 to see if PAR-2 remains present and localized to the posterior? This experiment could help clarify the discrepancy between the phenotypes and further support the role of SDS-22 in regulating GSP-2 protein levels. Additionally, by titrating PP1, the authors may be able to determine the minimum amount of PP1 needed to establish the PAR-2 domain.

    We will try this experiment but, assuming we find a condition in which we can fully deplete GSP-1 and only half of GSP-2, one problem is that it is impossible to control the levels of both GSP-1 and 2 and measure the PAR-2 domain in the same embryos (which would be the most rigorous way to perform the experiment so that we know the amount of depletion and correlate with the PAR-2 domain length). The only thing we can do is the same depletion time in the 3 different strains (the mNG::gsp-2, the gfp::gsp-1 and the gfp::par-2) and assume that the depletion will work the same in the three different strains.

    Minor comments

    Reviewer 1

    Minor Points

    • The link between lethality and polarity of the zygote is not always obvious and whether they are connected (or not) could probably be made clearer. Indeed, the source of lethality is unclear, particularly given that loss of SDS-22 on its own strongly impacts lethality with minimal effects on polarity (at least in the zygote).

    In many cases, we have reported embryonic lethality as information, not with a precise scope to correlate the lethality with the phenotype. We apologize for the lack of clarity. We know that embryonic lethality is normally associated with severe polarity defects. As example, in the par-2(RAFA) mutant and in the pkc-3ts mutant at temperatures around 24-25°C cortical polarity is lost, embryos divide symmetrically and synchronously and die (Calvi* et al., 2022; Rodriguez et al, 2017) and many more references for the PAR mutants (Kemphues et al, 1988; Kirby et al, 1990; Morton et al, 1992). We and others have also shown that depletion of GSP-2 can rescue the lethality of pkc-3(ts) but only at a semipermissive temperature when there is still residual PKC-3 activity (Calvi et al., 2022; Fievet et al, 2013). As our aim was to identify the regulator of GSP-2, we tested the potential regulators by RNAi in the pkc-3(ts), with the assumptions that a regulator, similar to GSP-2, would rescue the pkc-3(ts) polarity defects and lethality. As it turns out, SDS-22 is not a canonical regulator of GSP-2. The partial rescue of the polarity defects is most likely the result of the fact that SDS-22 lowers the level of GSP-2. However, SDS-22 is probably involved in many other functions that involve GSP-1 and GSP-2 (as shown for example:(Beacham et al, 2022; Peel et al.*, 2017)) and it is embryonic lethal. We do not know, however, whether the embryonic lethality is the results of the sum of the various functions of SDS-22 or it is due to a specific function.

    To clarify it better, we have now explained the connection between polarity and lethality in the text,

    From Result Line 111-114, page 5:

    We first asked whether depletion of any of these three regulators suppress the embryonic lethality of pkc-3(ne4246); gfp::par-2 embryos at the semi-permissive temperature of 24°C (in which PKC-3 is partially active, temperature used in all experiments with the pkc-3(ne4246) mutant, unless otherwise stated), similar to depletion of the catalytic subunit GSP-2.

    To Results Line 111-117, page 5:

    *When the temperature sensitive mutant pkc-3(ne4246) is grown at semi-permissive temperature, the residual PKC-3 activity is not sufficient to exclude PAR-2 from the anterior cortex. These embryos cannot establish polarity and die. Depletion of the catalytic subunit GSP-2 in this strain suppresses PAR-2 mislocalization and the resulting polarity defects, thereby rescuing embryonic lethality. We first asked whether depletion of any of these three identified regulators suppresses the embryonic lethality of pkc-3(ne4246); gfp::par-2 embryos at the semi-permissive temperature of 24°C (temperature used in all experiments with the pkc-3(ne4246) mutant, unless otherwise stated) , similar to depletion of GSP-2. *

    From Result Line 241-242, page 10:

    We next asked whether sds-22(E153A) was able to rescue the lethality and the polarity defects of *pkc-3(ne4246) *embryos.

    To Results Line 223-224, page 9:

    Because of this, we decided to test whether sds-22(E153A) was able to rescue the lethality and the polarity defects of pkc-3(ne4246) embryos.

    • Formally, the conclusion that reduced GSP-1/2 in SDS-22 depletion conditions is due to increased proteasomal degradation is not shown directly as there is no data on rates just steady-state levels. We agree that the genetic data is strongly suggestive of this model and it is consistent with work of other labs. Thus this is the most likely scenario, but could in principle reflect reduced expression that is balanced by reduced degradation.

    We agree with the reviewer. To address this point, we will perform RT-PCR analysis to measure the gene expression levels of gsp-1 and gsp-2 from control, SDS-22 depletion and *sds-22(E153A) *embryos.

    • It is interesting that sds-22(E153A) caused a stronger decrease in oocyte GSP-1 levels than sds-22(RNAi) (Fig 7). The authors may want to comment on this result.

    As we performed depletion of SDS-22 by RNAi feeding from L4 stage, we might not see strong reduction of GSP-1 in oocytes compared to that in sds-22(E153A) mutant, which carries an endogenous mutation of SDS-22 throughout the life cycle.

    Left, Representative images of* gfp::gsp-1* germlines in *ctrl(RNAi) *and *sds-22(RNAi), *comparing to gfp::gsp-1; sds-22(E153A); ctrl(RNAi). __Right, __Quantification of GFP::GSP-1 intensity levels in the cytoplasm and nucleus of -1 and -2 oocytes. N = 1.

    To address this point we have performed an experiment where we have depleted SDS-22 starting from L1s. As shown above, RNAi feeding of SDS-22 from L1 stage showed a similar reduction of GSP-1 (16.1% in the cytoplasm; 24.6% in the nucleus) as in gfp::gsp-1; sds-22(E153A), which was stronger comparing to feeding from L4 (8.8% in the cytoplasm; 17.4% in the nucleus, Fig 7D-E). This supports our hypothesis that the difference shown in Fig 7D-I might result from a relative short RNAi depletion of SDS-22 from L4 stage comparing to endogenous SDS-22(E153A) mutation. This experiment was done only once and will be repeated. If confirmed, we will add a sentence in the text. As RNAi feeding of SDS-22 from L1 stage impairs the formation of germlines, we will keep the protocol using SDS-22 RNAi feeding in L4 worms for other experiments in this study.

    • "At polarity establishment, the PP1 phosphatases GSP-1/-2 dephosphorylate PAR-2 allowing its cortical posterior accumulation." This statement, possibly inadvertently, implies temporal regulation, which has not been shown.

    We have changed the sentence, as suggested by the reviewer:

    To Introduction Line 59-60, page 3:

    The PP1 phosphatases GSP-1/-2 dephosphorylate PAR 2 allowing its cortical posterior accumulation and embryo polarization.

    • It would be ideal if the authors could explicitly state how they define pronuclear meeting. For example in Figure 1B, the embryos look like they are a few minutes past pronuclear meeting (e.g. compared to Figure 3), but maybe the pronuclei tend to meet more centrally in these conditions? Given that PAR-2 clearance is changing in time in some of these cases (based on looking at the movies), staging needs to be very accurate to get the best comparisons.

    We apologize for the lack of clarity. Pronuclear meeting is defined when the two pronuclei first contact each other.

    As noted by Reviewer 1, it is true that the pronuclei in pkc-3ts mutant tend to meet more centrally compared to control embryos. The same finding was also observed on PKC-3 inhibition (through depletion, mutation or inhibitor treatment) by Rodriguez et al (Rodriguez* et al., 2017). In addition, Kirby et al reported that mutations in the anterior PAR complex lead to the mislocalization of the pronuclei, causing them to meet more in the center (Kirby et al.*, 1990). We now specify this in the Material and Methods.

    Add in Material and Methods Line 633-635, page 22:

    *The stage of pronuclear meeting is defined when the two pronuclei first contact each other. In pkc-3(ne4246) embryos, the two pronuclei exhibited a tendency to meet more centrally compared to controls (Fig 1B, Movie EV1), as shown in (Kirby et al, 1990; Rodriguez et al, 2017). *

    As Reviewer 1 mentioned, accurate staging is crucial, as PAR-2 clearance can vary over time. The measurements were done in the first frame where pronuclei touch each other. However, in Fig. 1B we had shown one pkc-3ts; sds-22(RNAi) embryo one frame (10 seconds) later. We have now corrected this (see the updated Figure 1B).

    • In the interests of data-availability, upon publication the authors would deposit the raw mass spec data underlying Figure EV1.

    The reviewer is right, this was forgotten. We have now added as supplementary material the Dataset EV1 and EV2.

    Reviewer 3

    Minor comments: important issues that can confidently be addressed

    In the introduction (line 83), it's unclear what reconciles the contradictory data. I also have difficulty understanding this point in the discussion (line 435).

    We apologize for the lack of clarity and have now modified the text:

    From Introduction Line 82-84, page 4:

    This underscores the complex roles of SDS22 in regulating PP1 function and reconciling the contradictory data obtained in vivo and in vitro (Cao* et al., 2024; Cao et al, 2022; Kueck et al., 2024; Lesage et al*, 2007).

    To Introduction Line 81-85, page 4:

    These two recent findings suggest that while SDS-22 is required for the biogenesis of PP1 holoenzymes, its removal is essential to have an active PP1. This dual role of SDS-22 explains how SDS22 behaves as an inhibitor in biochemical assays in vitro but as an activator in vivo (Cao et al., 2024; Cao et al, 2022; Kueck et al., 2024; Lesage et al, 2007).

    From Discussion Line 435-436, page 17:

    These data reconcile the contradictory in vivo and in vitro observations.

    To Discussion Line 447-451, page 17:

    Given that SDS-22 both stabilizes PP1 levels and inhibits its activity, this dual role clarifies the apparent contradiction: while SDS-22 is essential for PP1 activity in vivo (because it is essential for the biogenesis/stability), it inhibits PP1 activity in vitro (as it needs to be removed to have an active PP1), while in vivo it is removed by p97/Valosin resulting in active PP1.

    Additionally, in the results section (line 389), it's not clear why the gonads cannot be studied in the strain with dead embryos. Are the gonads also altered in a way that prevents their observation?

    We explained this in the material and methods part (Line 583-584, 588-592), page 21.

    To clarify it better in the main text, we have now modified

    Results Line 377-378, page 15:

    Since depletion of these subunits results in worms with very little to no progeny (Fernando et al., 2022)

    Results Line 396-401, page 15:

    *Since we use the embryonic lethality phenotype of the mNG::gsp-2; sds-22(E153A) strain to recognize the homozygote sds-22(E153A), this precluded the possibility to analyze the germlines of homozygote mNG::gsp-2; sds-22(E153A) worms depleted of RNP-6.1 or RPN-7, as these worms do not have progenies (Fernando et al., 2022) and we therefore cannot distinguish the sds-22(E153A) homozygote from the sds-22(E153A) heterozygote (see material and methods for details). *

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    We have re-quantified the data in Fig 1B and displayed as in Fig 1C.

    We have double checked our data and corrected Fig 3G.

    We have modified the text to address many of the comments of the reviewer about clarity and rigor.

    We have added supplementary information Fig EV2C and Dataset EV1 and EV2.

    Other experiments performed are still preliminary and only shown in this revision letter.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    We believe with the reply, the text changes and the experiments that we have proposed and started, we will address all comments of the reiewers.

    References

    Beacham GM, Wei DT, Beyrent E, Zhang Y, Zheng J, Camacho MMK, Florens L, Hollopeter G (2022) The Caenorhabditis elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator. Genetics 222

    Calvi I, Schwager F, Gotta M (2022) PP1 phosphatases control PAR-2 localization and polarity establishment in C. elegans embryos. J Cell Biol 221

    Chartier NT, Salazar Ospina DP, Benkemoun L, Mayer M, Grill SW, Maddox AS, Labbe JC (2011) PAR-4/LKB1 mobilizes nonmuscle myosin through anillin to regulate C. elegans embryonic polarization and cytokinesis. Curr Biol 21: 259-269

    Fernando LM, Quesada-Candela C, Murray M, Ugoaru C, Yanowitz JL, Allen AK (2022) Proteasomal subunit depletions differentially affect germline integrity in C. elegans. Front Cell Dev Biol 10: 901320

    Fievet BT, Rodriguez J, Naganathan S, Lee C, Zeiser E, Ishidate T, Shirayama M, Grill S, Ahringer J (2013) Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy. Nat Cell Biol 15: 103-112

    Hao Y, Boyd L, Seydoux G (2006) Stabilization of cell polarity by the C. elegans RING protein PAR-2. Dev Cell 10: 199-208

    Hubatsch L, Peglion F, Reich JD, Rodrigues NT, Hirani N, Illukkumbura R, Goehring NW (2019) A cell size threshold limits cell polarity and asymmetric division potential. Nat Phys 15: 1075-1085

    Kemphues KJ, Priess JR, Morton DG, Cheng NS (1988) Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell 52: 311-320

    Kirby C, Kusch M, Kemphues K (1990) Mutations in the par genes of Caenorhabditis elegans affect cytoplasmic reorganization during the first cell cycle. Dev Biol 142: 203-215

    Klinkert K, Levernier N, Gross P, Gentili C, von Tobel L, Pierron M, Busso C, Herrman S, Grill SW, Kruse K* et al* (2018) Aurora A depletion reveals centrosome-independent polarization mechanism in C.elegans. bioRxiv: 388918

    Morton DG, Roos JM, Kemphues KJ (1992) par-4, a gene required for cytoplasmic localization and determination of specific cell types in Caenorhabditis elegans embryogenesis. Genetics 130: 771-790

    Park SH, Cheong C, Idoyaga J, Kim JY, Choi JH, Do Y, Lee H, Jo JH, Oh YS, Im W* et al* (2008) Generation and application of new rat monoclonal antibodies against synthetic FLAG and OLLAS tags for improved immunodetection. J Immunol Methods 331: 27-38

    Peel N, Iyer J, Naik A, Dougherty MP, Decker M, O'Connell KF (2017) Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication. PLoS Genet 13: e1006543

    Rodriguez J, Peglion F, Martin J, Hubatsch L, Reich J, Hirani N, Gubieda AG, Roffey J, Fernandes AR, St Johnston D* et al* (2017) aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity. Dev Cell 42: 400-415 e409

    Shimada M, Kanematsu K, Tanaka K, Yokosawa H, Kawahara H (2006) Proteasomal ubiquitin receptor RPN-10 controls sex determination in Caenorhabditis elegans. Mol Biol Cell 17: 5356-5371

    Tzur YB, Egydio de Carvalho C, Nadarajan S, Van Bostelen I, Gu Y, Chu DS, Cheeseman IM, Colaiacovo MP (2012) LAB-1 targets PP1 and restricts Aurora B kinase upon entrance into meiosis to promote sister chromatid cohesion. PLoS Biol 10: e1001378

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary: your understanding of the study and its conclusions

    This is a follow up on Gotta's lab paper, which shows that the PP1 catalytic subunits GSP-1 and GSP-2 are involved in the polarization of the C. elegans zygote (10.1083/jcb.202201048). Here, the authors report that SDS-22, an interactor of PP1, regulates PP1 function in the zygote. Depleting SDS-22, similar to depleting GSP-2, rescues the polarity defects caused by the inactivation of aPKC in the zygote. This suggests that SDS-22 plays a role in promoting GSP-2's function in polarity. The mechanism behind this may involve SDS-22 protecting GSP-1 and GSP-2 from degradation by the proteasome.

    Major comments: major issues affecting the conclusions

    Overall, the authors' conclusions are supported by their data. The data and methods are presented clearly, with appropriate replicates and statistics. Here I propose two experiments to strengthen the link between some of their data and their claims. These experiments could take a month or two to complete.

    Experiment 1

    It would be helpful if the authors could show that blocking the proteasome in the zygote restores GSP-1/-2 levels in the absence of SDS-22 or even better in the SDS-22(E153A) mutant. This would provide more direct evidence to support their claim that SDS-22 regulates polarity by protecting PP1 from proteasomal degradation. While they are currently conducting this experiment in the germline, they cannot assess polarity there. However, in the zygote, they would be able to examine the PAR-2 domain (polarity). To do this, the authors could permeabilise the embryos and apply a proteasome inhibitor.

    Experiment 2

    The posterior localization of PAR-2 after co-RNAi of GSP-1 and SDS-22 contrasts with the absence of PAR-2 at the cortex when both GSP-1 and GSP-2 are depleted. This difference may be due to the partial reduction of GSP-2 levels when SDS-22 is depleted, compared to the more substantial reduction of GSP-2 upon GSP-2 RNAi. Have the authors considered combining full depletion of GSP-1 with partial depletion of GSP-2 to see if PAR-2 remains present and localized to the posterior? This experiment could help clarify the discrepancy between the phenotypes and further support the role of SDS-22 in regulating GSP-2 protein levels. Additionally, by titrating PP1, the authors may be able to determine the minimum amount of PP1 needed to establish the PAR-2 domain.

    Minor comments: important issues that can confidently be addressed

    In the introduction (line 83), it's unclear what reconciles the contradictory data. I also have difficulty understanding this point in the discussion (line 435). Additionally, in the results section (line 389), it's not clear why the gonads cannot be studied in the strain with dead embryos. Are the gonads also altered in a way that prevents their observation?

    Referees cross-commenting

    Overall, I agree with the other reviewers' comments. The suggested experiments would help strengthen the connection between SDS-22 and cell polarity, as well as its role in relation to the proteasomal-mediated degradation of GSP-1/-2 and its impact on cell polarity. These experiments seem feasible and could provide stronger support for the authors' claims about these regulatory mechanisms. Alternatively, the authors may consider moderating some of their conclusions if these experiments are not conducted.

    Significance

    General assessment: strengths and limitations

    This study enhances our understanding of how phosphatases regulate cell polarity, specifically in the C. elegans zygote, a key model system for studying cell polarity. The study could be further strengthened by the experiments mentioned above. Additionally, see the comment on how to increase the impact of the work (Audience section).

    Advance: compare the study to existing published knowledge This study is the first to characterize the role of SDS-22 in the polarization of the C. elegans zygote. As the authors discuss, their results align with and complement existing knowledge of SDS-22 in other cell types. Together with the literature, this work highlights the complexity of PP1 regulation, suggesting that different PP1 outcomes may be achieved by combining SDS-22 with various PP1 co-regulators.

    Audience that will be interested or influenced by this research

    These results will be of interest to scientists studying cell signalling and cell polarity. There is currently strong focus on understanding the regulation of phosphatases. In cell polarity research, the spatial regulation of phosphatases is particularly important for understanding the asymmetric activation of signalling pathways. SDS-22 does not appear to control the spatial localization or activity of PP1, but rather its overall protein levels. As the authors note in the discussion, this suggests that other factors may be involved in the polarization of PP1 signalling. In supplementary figure S1, the authors provide a volcano plot showing candidate PP1 interactors. Providing the list of positive hits would increase the impact of the study and benefit the research community. It would also help explain why the authors chose to follow up on SDS-22 in this study. Furthermore, this could advance the identification of factors involved in the polarization of PP1 signalling.

    My expertise

    Cell polarity, cell signalling, embryo development.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary: The authors present a logical and clear set of data that support the model that SDS-22 is an important regulator of cell polarity via its ability to stabilize Protein Phosphatase 1 (PP1).

    The authors use a clever combination of genetic manipulations and quantitative imaging to show that loss of SDS-22 phenocopies loss of PP1, in that PAR-2 polarization is restored in nascent C. elegans zygotes following inactivation of PKC-3. Rescue of PAR-2 polarization also occurs when the authors mutate a conserved residue in SDS-2 that is predicted to form electrostatic interactions with PP1, suggesting that SDS-22 acts via PP1. Inactivation of SDS-22 results in decreased levels of PP1, and the authors provide evidence that this is via proteasomal degradation by showing that PP1 levels are restored by knockdown of proteasomal subunits.

    Overall the manuscript is well-written, the experiments rigorous, and the methods and data likely to be reproducible.

    Major comment: Consistent with the model that PP1 activity is reduced in the absence of SDS-22, the authors show that a surrogate PP1 target (phospho-histone H3) becomes hyper-phosphorylated. To strengthen the study, the authors could consider performing an OPTIONAL experiment (see below) of assaying the phosphorylation status of PAR-2 itself, as this is proposed to be the target of both PKC-3 and PP1, and represent the mechanism of PAR-2 polarization.

    Referees cross-commenting

    In principle I agree with many of the thoughtful comments by the other reviewers. They point out many potential areas for both enhancing the strength of the findings and including a more nuanced interpretation of the results. However, I also feel that the experiments proposed to deal with their concerns might not be so straightforward to pursue for unforeseen technical reasons and may actually take substantially longer than anticipated. The same is true for my proposed experiment to assess phosphorylation status of PAR-2, which is why I have indicated it as optional. I ask the other reviewers to consider if any of their proposed experiments might also be considered optional. I also thank them for their critical assessments of the paper! They were helpful for me and I'm sure will also be for the authors.

    Significance

    This study brings clarity to the contentious role of SDS-22 by showing that it appears to promote PP1 activity by counteracting the phosphatase degradation process in vivo. This complements a previously hypothesized function of SDS-22, while contrasting with other proposed functions of SDS-22 as a regulator of PP1 localization or stimulator of PP1 degradation. Thus, the authors' discoveries in C. elegans represent a significant advance in our understanding of protein phosphatase regulation, a long-standing question in biology and a central process in all cellular systems. The study also points to potential mechanisms for modulating phosphatase activity in other contexts, across different organisms and disease states. Basic science researchers will be interested in the findings, with potential to attract additional interest from physiologists and even drug designers.

    One limitation of the study is that the authors use PAR-2 polarization as a readout of PKC-3 and PP1 activity without showing directly that PAR-2 phosphorylation status is changing in response to their genetic manipulations, including SDS-22 inactivation. The PAR-2 membrane localization is thought to be inhibited by PKC-3-dependent phosphorylation and promoted by PP1-dependent dephosphorylation. Is there a possibility of examining whether PAR-2 phosphorylation is elevated in SDS-22 RNAi or mutant animals? Previously, Hao et. al., 2006; doi.org/10.1016/j.devcel.2005.12.015. showed in Figure 2 that PAR-2 runs as a doublet band on western blots with the phosphorylated form of PAR-2 appearing to correlate with the slightly higher molecular weight band. This was used to infer the ratio of phosphorylated to dephosphorylated PAR-2. I'm wondering if it might be possible for the authors to perform a similar analysis of their existing GFP::PAR-2? It appears from their previous paper on PP1 regulation of PAR-2 polarization (Calvi et. al., 2022; doi: 10.1083/jcb.202201048.) that they might also be detecting a similar doublet (Figure S5F and associated source file), so perhaps it is a doable experiment? Regardless, this is not an essential experiment as the study is already significant and rigorous!

    I am a cell biologist that uses C. elegans to understand the function of conserved protein complexes that regulate the development and function of animal tissues.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary

    This work from Li et al. identifies a role for SDS-22 in maintaining normal levels of the PP1 subunits GSP-1/2 in C. elegans. Prior work in the lab identified a role for the catalytic PP1 subunits GSP-1/2 in opposing the phosphorylation of of the polarity protein PAR-2 by the polarity kinase PKC-3(aPKC). To identify potential regulatory subunits in this process, they performed IP-MassSpec on GSP-2 and pulled out a number of potential regulatory subunits, including SDS-22. While polarity was the primary motivation for the study, their subsequent analysis did not point to a specific function in cell polarity, but rather pointed to a general effect on stabilising levels of GSP-1/2 against potential proteasome-mediated degradation. Consistent with this hypothesis, SDS-22 depletion or mutation of the SDS-22-GSP-1/2 interaction partially recapitulated the phenotypes of GSP-1/2 depletion including increased phosphorylation of histone H3. Consistent with reduced PP1 activity, there were modest effects on polarity as seen in GSP-2 depleted embryos, including slightly reducing PAR-2 domain size and partially restoring PAR-2 asymmetry in embryos carrying a temperature sensitive pkc-3 mutation.

    Major Comments:

    1. Overall, the evidence supporting the core finding that SDS-22 is required for normal GSP-1/2 levels is strong and well documented. The experiments were performed well and controls, statistics, replicates were appropriate. Our only slight reservation was whether the effect of sds-22(RNAi) on stability may be overstated due to the use of GFP fusions to GSP-1/2 for this analysis. The authors note these alleles are hypomorphic, potentially raising the possibility that GFP tags destabilise the proteins and make them more prone to degradation. Ideally this would be repeated with an untagged allele via Western (e.g. Peel et al 2017 for relevant antibodies).
    2. The role for SDS-22 in polarity is rather weak. Both the SDS-22 depletion phenotypes and the ability of SDS-22 depletion to suppress pkc-3(ts) polarity phenotypes are modest (and weaker in than GSP-2 depletion). For example, the images in Figure 1B appear striking, but from Movie S1 it is clear that this isn't a full rescue as PAR-2 is initially uniformly enriched on the cortex (rather than mostly cytoplasmic) and it is never fully cleared. In the movie, the clearance at the point of pronuclear meeting is very modest. Quantitation might be helpful here (i.e. as in Figure 3G). As the authors state, it seems that SDS-22 does not have a specific role in polarity beyond the general effect on GSP-1/2 levels. This does not undermine the core message of the paper, but we would recommend downplaying the conclusions with respect to contributing to polarity establishment. For example "...suggesting that SDS-22 regulates GSP-1/-2 activity to control the loading of PAR-2 to the posterior cortex in one-cell stage C. elegans embryos" implies a regulatory role for SDS-22 in polarity, but we would interpret it as simply helping reduce aberrant degradation of GSP-1/2 and this impacts a variety of cellular processes including polarity.
    3. Specificity of SDS-22 effects on polarity. SDS-22 (or GSP-1/2) depletion is likely to have effects on many pathways. We wondered whether some of the polarity phenotypes may not be specifically due to changes in the PAR-2 phosphorylation cycle as implied.

    One candidate is the actomyosin cortex. It was noticeable that control and sds-22 embryos were different: In Movies S1, S2, and S3 control embryos show either stronger or more persistent cortical ruffling or pseudocleavage furrows. This is also visible in Figure 3A. Is it possible that disruption of SDS-22 reduces cortical flows (time, intensity or duration) and could this explain the small reduction in anterior PAR-2 spreading and thus the slightly smaller domain size measured in Figures 1B and 3A.

    A potentially related issue could be embryo size. sds-22 embryos generally seem to be smaller than wild-type (e.g. Figure 1B(left), 4A(left column), and particularly EV3). Is this consistently true? Could cell size effects change the ability of embryos to clear anterior PAR-2 domains as described in EV3? Klinkert et al (2018, biorXiv) note that reducing the size of air-1(RNAi) embryos reduces the frequency of bipolar PAR-2 domains.

    We would stress that these comments relate to interpreting the polarity phenotypes and do not undermine the core finding that SDS-22 stabilises GSP-1/2.

    Minor Points

    • The link between lethality and polarity of the zygote is not always obvious and whether they are connected (or not) could probably be made clearer. Indeed, the source of lethality is unclear, particularly given that loss of SDS-22 on its own strongly impacts lethality with minimal effects on polarity (at least in the zygote).
    • Formally, the conclusion that reduced GSP-1/2 in SDS-22 depletion conditions is due to increased proteasomal degradation is not shown directly as there is no data on rates just steady-state levels. We agree that the genetic data is strongly suggestive of this model and it is consistent with work of other labs. Thus this is the most likely scenario, but could in principle reflect reduced expression that is balanced by reduced degradation.
    • It is interesting that sds-22(E153A) caused a stronger decrease in oocyte GSP-1 levels than sds-22(RNAi) (Fig 7). The authors may want to comment on this result.
    • "At polarity establishment, the PP1 phosphatases GSP-1/-2 dephosphorylate PAR-2 allowing its cortical posterior accumulation." This statement, possibly inadvertently, implies temporal regulation, which has not been shown.
    • It would be ideal if the authors could explicitly state how they define pronuclear meeting. For example in Figure 1B, the embryos look like they are a few minutes past pronuclear meeting (e.g. compared to Figure 3), but maybe the pronuclei tend to meet more centrally in these conditions? Given that PAR-2 clearance is changing in time in some of these cases (based on looking at the movies), staging needs to be very accurate to get the best comparisons.
    • In the interests of data-availability, upon publication the authors would deposit the raw mass spec data underlying Figure EV1.

    Referees cross-commenting

    We also generally agree with the comments of the other reviewers.

    Our only concern with the main conclusion regarding GSP stabilization of GSP-1/2 is the impact of the gfp tags. Given that antibodies exist and have been used in Peel et al 2017 for exactly this purpose in C. elegans embryos, this does not seem excessively burdensome in our view and would strengthen the paper.

    The remainder of our concerns can likely be addressed by modifications to the text and/or adding a caveats/limitations section to their discussion. As we noted, these mostly relate to the magnitude and specificity of the impact of SDS-22 on polarity and PAR-2 phosphorylation, which in our view is rather peripheral to the core conclusion (i.e. that SDS-22 stabilizes GSP-1/2).

    Significance

    Overall, this is a careful and well-executed study identifying a conserved role for SDS-22 in stabilising PP1 catalytic subunits in C. elegans embryos and shows that this can broadly impact PP1 activity in this system. A mechanistic role for SDS-22 in PP1 function was recently demonstrated in (Cao et al, 2024), where it was shown to stabilise nascent catalytic subunits, but also in subunit recycling (Kuetsch et al 2024). The data here suggest this role in stabilisation PP1 subunits is broadly relevant.

    These data are also consistent with prior work from the lab demonstrating the role of PP1 in C. elegans zygote polarity. It adds to previous reports that compromised PP1 activity can impact cell polarity and further highlights the importance of considering regulation of protein phosphatases in cell polarity pathways. That said, the impact on polarity is rather modest, likely reflecting a general requirement for SDS-22 in supporting optimal PP1 activity rather than any specific role in polarity.

    Field of expertise: cell polarity, cell and developmental biology.

  5. Version published to 10.1101/2025.01.07.631699 on bioRxiv