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  1. eLife

    Author Response

    Reviewer #1 (Public Review):

    Auxin-induced degradation is a strong tool to deplete CHK-2 and PLK-2 in the C. elegans germ line. The authors strengthen their conclusions through multiple approaches, including rescuing mutant phenotypes and biochemical analyses of CHK-2 and PLK-2.

    The authors overcame a technical limitation that would hinder in vitro analysis (low quantity of CHK-2) through the clever approach of preventing its degradation via the proteasome. In vitro phosphorylation assays and mass spectrometry analysis that establishes that CHK-2 is a substrate of PLK-2 nicely complement the genetic data.

    The authors argue that the inactivation of CHK-2 by PLK-2 promotes crossover designation; however, the data only indicate that PLK-2 promotes proper timing of crossover designation.

    We thank the reviewer for this point of clarification. While we believe that PLK activity is essential to inactivate CHK-2 and trigger CO designation, we agree that this has not been firmly established with the tools available to us, as elaborated below. We have revised the text to avoid overstating the conclusions.

    It is not clear whether the loss of CHK-2 function with the S116A and T120A mutations is the direct result of the inability to phosphorylate these residues or whether it is caused by the apparent instability of these proteins, as their abundance was reduced in IPs compared to wild-type. Agreed. The instability of the mutant proteins was a source of significant frustration during the course of this work, and limits the strength of our conclusions.

    The mechanism of CHK-2 inactivation in the absence of PLK-2 remains unclear, though the authors were able to rule out multiple candidates that could have played this role.

    Reviewer #2 (Public Review):

    In this manuscript, Zhang et al., address the role of Polo-like kinase signaling in restricting the activity of Chk2 kinase and coordinating synapsis among homologous chromosomes with the progression of meiotic prophase in C. elegans. While individual activities of PLK-2 and CHK-2 have been demonstrated to promote chromosome pairing, and double-strand break formation necessary for homologous recombination, in this manuscript the authors attempt to link the function of these two essential kinases to assess the requirement of CHK-2 activity in controlling crossover assurance and thus chromosome segregation. The study reveals that CHK-2 acts at distinct regions of the C. elegans germline in a Polo-like kinase-dependent and independent manner.

    Strengths:

    The study reveals distinct mechanisms through which CHK-2 functions in different spatial regions of meiosis. For example, it appears that CHK-2 activity is not inhibited by PLK's (1 and 2) in the leptotene/zygotene meiotic nuclei where pairing occurs. This suggests that either CHK-2 is not phosphorylated by PLK-2 in the distal nuclei or that it has a kinase-independent function in this spatial region of the germline. These are interesting observations that further our understanding of how the processes of meiosis are orchestrated spatially for coordinated regulation of the temporal process.

    Weaknesses:

    While the possibilities stated above are interesting, they lack direct support from the data. A key missing element in the study is the actual role of PLK-2 signaling in controlling CHK-2 activity and thus function. I expand on this below.

    Throughout the manuscript, the authors test the role of each of the kinases (CHK-2 or PLK-1, or 2) using auxin-induced degradation, which would eliminate both phosphorylated and unphosphorylated pools of proteins. This experiment thus does not test the role of PLK-2 signaling in controlling CHK-2 function or the role of CHK-2 activation. To test the role of signaling from PLK-2 or CHK-2, the authors need to generate appropriate alleles such as phospho-mutants or kinase-dead mutants. The authors do generate unphosphorylatable and phosphomimetic versions of CHK-2, however, they find that the protein level for both these alleles is lower than wild-type CHK-2 (which the authors state is already low). The authors conclude that the lower level of protein in the CHK-2 phospho-mutants is because the mutations cause destabilization of the protein. I am sympathetic with the authors since clearly these results make interpretations of actual signaling activity more challenging. But there needs to be some evidence of this activity, for example through the generation of a phosphor-specific antibody to phosphorylated CHK-2. While not functional, at least the phosphorylation status of CHK-2 would provide more information on its spatial pattern of activation and inactivation. In addition, it would still be of interest to the readership to present the data on these phosphor-mutant alleles with crossover designation and COSA-1::GFP. Is the phenotype of the WT knockin, and each of the phosphomutant knock-ins similar to auxin-induced degradation of CHK-2?

    We thank the reviewer for these comments. We have made several attempts over the past decade that have failed to elicit a CHK-2 antibody that works for either immunofluorescence or western blots, likely due to the very low abundance of CHK-2. This has discouraged us from investing yet more resources to try to develop a phospho-specific antibody. Moreover, our evidence suggests that phosphorylation may promote CHK-2 degradation. Since the phosphomutants of CHK-2 are not stable, we do not think knock-in of these phosphomutants will provide new insights.

    Given that the CHK-2 phosphomutants did not pan out for assessing the signaling regulation of PLK-2 on CHK-2, to directly assess whether PLK-2 activity restricts CHK-2 function in mid-pachytene but not leptotene/zygotene, the authors should generate PLK-2 kinase dead alleles. These alleles will help decouple the signaling function of PLK-2 from a structural function.

    Similarly, to assess the potentially distinct roles of CHK-2 in leptotene/zygotene and mid-pachytene it would be important to assess CHK-2 kinase-dead mutant alleles. At this time, all of the analysis is based on removing both active CHK-2 and inactive CHK-2 (i.e. phosphorylated and unphosphorylated pool) using auxin-induced degradation. The kinase-dead alleles will help infer the role of the kinase more directly. The authors can then superimpose the auxin-induced degradation and assess the impact of complete removal of the protein vs only loss of its kinase function. These experiments may help clarify the role of signaling outcomes of these proteins, vs their complete loss. For example, what does kinase dead PLK-2 recruitment to the synapsed chromosomes appear like? Are their distinct activities for active and inactive PLK-2 that are spatially regulated? The same can be tested for CHK-2.

    A kinase-dead allele of plk-2 has been generated in previous work and we have used it for other purposes. However, the fact that CHK-2 and PLK-2 are required for homolog pairing and synapsis, which are prerequisites for crossover designation, precludes their use here.

  2. eLife

    eLife assessment

    Zhang et al. present convincing data describing a role for Polo-like kinase PLK-2 in restricting the activity of Chk2 kinase and coordinating synapsis of homologous chromosomes with the progression of meiotic prophase in C. elegans. By revealing PLK-2-dependent and -independent mechanisms of CHK-2 activity, this work provides a valuable understanding of the major regulators of meiotic progression.

  3. eLife

    Reviewer #1 (Public Review):

    Auxin-induced degradation is a strong tool to deplete CHK-2 and PLK-2 in the C. elegans germ line. The authors strengthen their conclusions through multiple approaches, including rescuing mutant phenotypes and biochemical analyses of CHK-2 and PLK-2.

    The authors overcame a technical limitation that would hinder in vitro analysis (low quantity of CHK-2) through the clever approach of preventing its degradation via the proteasome. In vitro phosphorylation assays and mass spectrometry analysis that establishes that CHK-2 is a substrate of PLK-2 nicely complement the genetic data.

    The authors argue that the inactivation of CHK-2 by PLK-2 promotes crossover designation; however, the data only indicate that PLK-2 promotes proper timing of crossover designation.

    It is not clear whether the loss of CHK-2 function with the S116A and T120A mutations is the direct result of the inability to phosphorylate these residues or whether it is caused by the apparent instability of these proteins, as their abundance was reduced in IPs compared to wild-type.

    The mechanism of CHK-2 inactivation in the absence of PLK-2 remains unclear, though the authors were able to rule out multiple candidates that could have played this role.

  4. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Zhang et al., address the role of Polo-like kinase signaling in restricting the activity of Chk2 kinase and coordinating synapsis among homologous chromosomes with the progression of meiotic prophase in C. elegans. While individual activities of PLK-2 and CHK-2 have been demonstrated to promote chromosome pairing, and double-strand break formation necessary for homologous recombination, in this manuscript the authors attempt to link the function of these two essential kinases to assess the requirement of CHK-2 activity in controlling crossover assurance and thus chromosome segregation. The study reveals that CHK-2 acts at distinct regions of the C. elegans germline in a Polo-like kinase-dependent and independent manner.

    Strengths:
    The study reveals distinct mechanisms through which CHK-2 functions in different spatial regions of meiosis. For example, it appears that CHK-2 activity is not inhibited by PLK's (1 and 2) in the leptotene/zygotene meiotic nuclei where pairing occurs. This suggests that either CHK-2 is not phosphorylated by PLK-2 in the distal nuclei or that it has a kinase-independent function in this spatial region of the germline. These are interesting observations that further our understanding of how the processes of meiosis are orchestrated spatially for coordinated regulation of the temporal process.

    Weaknesses:
    While the possibilities stated above are interesting, they lack direct support from the data. A key missing element in the study is the actual role of PLK-2 signaling in controlling CHK-2 activity and thus function. I expand on this below.

    Throughout the manuscript, the authors test the role of each of the kinases (CHK-2 or PLK-1, or 2) using auxin-induced degradation, which would eliminate both phosphorylated and unphosphorylated pools of proteins. This experiment thus does not test the role of PLK-2 signaling in controlling CHK-2 function or the role of CHK-2 activation. To test the role of signaling from PLK-2 or CHK-2, the authors need to generate appropriate alleles such as phospho-mutants or kinase-dead mutants. The authors do generate unphosphorylatable and phosphomimetic versions of CHK-2, however, they find that the protein level for both these alleles is lower than wild-type CHK-2 (which the authors state is already low). The authors conclude that the lower level of protein in the CHK-2 phospho-mutants is because the mutations cause destabilization of the protein. I am sympathetic with the authors since clearly these results make interpretations of actual signaling activity more challenging. But there needs to be some evidence of this activity, for example through the generation of a phosphor-specific antibody to phosphorylated CHK-2. While not functional, at least the phosphorylation status of CHK-2 would provide more information on its spatial pattern of activation and inactivation. In addition, it would still be of interest to the readership to present the data on these phosphor-mutant alleles with crossover designation and COSA-1::GFP. Is the phenotype of the WT knockin, and each of the phosphomutant knock-ins similar to auxin-induced degradation of CHK-2?

    Given that the CHK-2 phosphomutants did not pan out for assessing the signaling regulation of PLK-2 on CHK-2, to directly assess whether PLK-2 activity restricts CHK-2 function in mid-pachytene but not leptotene/zygotene, the authors should generate PLK-2 kinase dead alleles. These alleles will help decouple the signaling function of PLK-2 from a structural function.

    Similarly, to assess the potentially distinct roles of CHK-2 in leptotene/zygotene and mid-pachytene it would be important to assess CHK-2 kinase-dead mutant alleles. At this time, all of the analysis is based on removing both active CHK-2 and inactive CHK-2 (i.e. phosphorylated and unphosphorylated pool) using auxin-induced degradation. The kinase-dead alleles will help infer the role of the kinase more directly. The authors can then superimpose the auxin-induced degradation and assess the impact of complete removal of the protein vs only loss of its kinase function. These experiments may help clarify the role of signaling outcomes of these proteins, vs their complete loss. For example, what does kinase dead PLK-2 recruitment to the synapsed chromosomes appear like? Are their distinct activities for active and inactive PLK-2 that are spatially regulated? The same can be tested for CHK-2.

  5. eLife

    Reviewer #3 (Public Review):

    In this paper, Zhang et al. investigated the regulation of the meiotic checkpoint kinase CHK-2, whose inactivation is a necessary step in ensuring that chromosomes have synapsed and received crossovers before progression to later events of meiotic prophase. Using mass spectrometry, biochemistry, and cytological analysis of mutant and transgenic strains, they show that CHK-2 is phosphorylated and that CHK-2 activity is attenuated in a manner dependent on recruitment of the kinase PLK-2 to a conserved docking motif on the synaptonemal complex, which forms between pairs of homologous chromosomes. The results plausibly explain how CHK-2 can remain active and prolong the events of early prophase chromosome dynamics in response to delays in synapsis since unsynapsed chromosomes will not recruit PLK-2 to inactivate CHK-2 locally. While molecular details remain to be worked out (e.g., why the loss of crossover intermediates can also extend CHK-2 activity; why PLK-2 does not inactivate CHK-2 at pairing centers), this work provides an elegant explanatory unification of several disparate observations.

    The authors made extensive use of the auxin-inducible degron system combined with the spatiotemporal arrangement of the C. elegans germline to examine the effect of conditional depletion of proteins in cells where the depleted protein was required for earlier events. This is a powerful approach that can give stronger evidence than an examination of genetic mutant backgrounds, especially when, as in this paper, controls are performed to confirm the timing of depletion by loss of immunofluorescence signal. The method of measuring the proportion of the gonad occupied by nuclei with bright COSA-1 foci is generally robust, but the criteria for demarcation could be more strictly defined. For example, does a single nucleus with a single bright COSA-1 spot suffice to mark the beginning of a zone?

    A weakness of this paper is that the non-phosphorylatable alleles constructed to provide a functional test of CHK-2 phosphorylation, unfortunately, had severe meiotic defects, so the importance of CHK-2 phosphorylation in its deactivation remains uncertain. While the results overall point towards direct phosphorylation of CHK-2 by PLK-2 (and possibly PLK-1), the authors are careful to point out that this is not the only possible explanation. In this regard, the mass spectrometry data should be given a statistical analysis to see whether they are best explained by in vitro phosphorylation of CHK-2 by PLK-2.

  6. Version 2 published on bioRxiv
  7. Version 1 published on bioRxiv