BUB-1 and CENP-C recruit PLK-1 to control chromosome alignment and segregation during meiosis I in C. elegans oocytes

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1. 

   Version published to 10.7554/elife.84057 on eLife

   Apr 17, 2023
2. 

   eLife

   Mar 31, 2023

   Author Response

   Reviewer #1 (Public Review):

   This study focuses on the role of polo like kinase 1 (PLK-1) during oocyte meiosis. In mammalian oocytes, Plk1 localizes to chromosomes and spindle poles, and there is evidence that it is required for nuclear envelope breakdown, spindle formation, chromosome segregation, and polar body extrusion. However, how Plk1 is targeted to its various locations and how it performs these functions is not well understood. This study uses C. elegans oocytes as a model to explore PLK-1 function during meiosis. They take advantage of an analogue-sensitive allele of plk-1, which enabled them to bypass nuclear envelope breakdown defects that occur following PLK-1 RNAi. This allowed them to dissect later roles of PLK-1 in oocytes, demonstrating that depletion causes defects in spindle organization, …

   Author Response

   Reviewer #1 (Public Review):

   This study focuses on the role of polo like kinase 1 (PLK-1) during oocyte meiosis. In mammalian oocytes, Plk1 localizes to chromosomes and spindle poles, and there is evidence that it is required for nuclear envelope breakdown, spindle formation, chromosome segregation, and polar body extrusion. However, how Plk1 is targeted to its various locations and how it performs these functions is not well understood. This study uses C. elegans oocytes as a model to explore PLK-1 function during meiosis. They take advantage of an analogue-sensitive allele of plk-1, which enabled them to bypass nuclear envelope breakdown defects that occur following PLK-1 RNAi. This allowed them to dissect later roles of PLK-1 in oocytes, demonstrating that depletion causes defects in spindle organization, chromosome congression, segregation, and polar body extrusion. Moreover, the authors defined mechanisms by which PLK-1 is targeted to chromosomes, showing that CENP-C (HCP-4) is required for localization to chromosome arms and that BUB-1 is required for targeting to the midbivalent region. Finally, they demonstrate that upon removal of PLK-1 from both domains, there are severe meiotic defects. These findings are interesting. However, there is a need for additional analysis to better support some of their conclusions, and to aid in interpretation of particular phenotypes. Specific comments are below.

   • For many important claims of the paper, a single representative image is shown but the n is not noted. This is an issue throughout the paper for much of the localization analysis (e.g. Figure 1B, 1C, 1D, 2A, 2B, 3A, 3B, 3C, etc.); in cases like this, numbers should be included to increase the rigor of the presented data. How many images or movies were analyzed that looked like the one shown? For linescans, were they done only on one image? How many independent experiments were done, etc?

   We had initially chosen a representative image. Localisation was the same in all images that allowed ‘proper’ assessment of PLK-1 localisation. In our case, this means that we can only analyse bivalents that are perpendicular to the light path to distinguish between bivalent, chromosome arms, and kinetochore. We now report the number of oocytes (N) and bivalents (n) analysed for each condition. The line scans were done in one representative image.

   • In the abstract, it is stated that PLK-1 plays a role in spindle assembly/stability (this is also stated elsewhere, e.g. line 101). This phrasing implies that the authors have demonstrated roles in both spindle assembly and stability. However, to distinguish between these roles, they would have to show that removal of PLK-1 before spindle assembly causes defects, and also that removal of PLK-1 from pre-formed spindles causes collapse. I don't think it is necessary to do this, as the spindle roles of PLK-1 are not a focus of the paper. However, the language should be altered so that it does not imply that the paper has demonstrated roles in both. A good place to do this would be in the section from lines 144-147, where they first discuss the spindle defects. It would be straightforward to explain that their approach does not distinguish between spindle assembly and stability, and that PLK-1 could have a role in either or both.

   We fully agree with this comment. We cannot distinguish between spindle assembly and stability, and it is also not the focus of our current work. We have changed the text accordingly.

   • It is stated that there is kinetochore localization of PLK-1 (and I do see some dim cup-like localization in images after PLK-1 is removed from the chromosome arms via HCP-4 RNAi). However, this cup-like localization is not clear in most wild-type images (e.g. Figure 1B, 1D, 2A, 3A, etc.). Although I recognize that the chromatin staining might be obscuring kinetochore localization, if PLK-1 was truly a kinetochore protein I would also expect it to localize to filaments within the spindle (as many other kinetochore proteins do), especially since the authors state that BUB-1 targets PLK-1 to the kinetochore (and BUB-1 is in the filaments). In fact, the only images where it looks like PLK-1 may be localized to filaments are in Figure 4C and 6A, when HCP-4 has been depleted (though I don't know if this generally true across all HCP-4 RNAi images). For me, this calls into question the conclusion that PLK-1 truly is on the kinetochore in wild type conditions - could it be that PLK-1 only localizes to the kinetochore (and to the filaments) when HCP-4 is depleted? The authors need to resolve this issue and provide better evidence that PLK-1 normally localizes to the kinetochore, if they want to make this claim. Additionally, the observation that PLK-1 is not on the kinetochore filaments (in wild type conditions) should be addressed in the text somewhere - do the authors think that this is a special type of kinetochore protein that does not localize to the filaments?

   While our initial claim of PLK-1 kinetochore localisation was based on its cup-like localisation, we have now performed additional analysis and experiments to confirm this claim. First, we corroborated that PLK-1 cup-like pattern co-localises with the Mis12 complex component KNL-3 (New Figure 5-figure supplement 1). Second, we show that PLK-1 is present in the so called ‘linear elements’ (filaments) both within the spindle and in the cortex. Since PLK-1 presence in these filaments is seen in wild type as well as hcp-4 mutant oocytes, we conclude that PLK-1 likely localises in kinetochore in normal conditions.

   • The authors should provide a control experiment, treating wild-type worms with 10uM 3-IB-PP1. This would be important to ensure that the spindle defects seen at this concentration in the plk-1as strain are not non-specific effects of the inhibitor. There is a control in Figure 1 - figure supplement 3 using 1uM 3-IB-PP1 but didn't see a control for 10uM (the concentration at which spindle defects are observed).

   This control has now been included in Figure 1-figure supplement 3.

   • In Figure 2F, the gels for BUB-1+PLK-1 look different in the presence and absence of phosphorylation by Cdk1 - for these data, I agree with the authors that it looks as if the complex elutes at a higher volume if BUB-1 is not phosphorylated (lines 200-204). However, Figure 2G has a repeat of the condition with phosphorylated BUB-1, and in this panel, the complex appears to elute at a higher volume than it did on the gel in panel F. The gel in panel G looks much more similar to the unphosphorylated condition in panel F. The authors need to explain this discrepancy (i.e., Is there a reason why the gels cannot be compared between panels? How reproducible are these data?). Ideally, the authors would include a repeat of the unphosphorylated BUB-1 + PLK-1 condition in panel G, done at the same time as the conditions shown in that panel, to avoid the impression that their results may not be reproducible.

   The specific elution volume cannot be compared in different experiments as the column has proven to “drift” over time – with proteins eluting at a later volume than they did previously despite extensive washing. What is reproducible under the experimental conditions is that the unphosphorylated wild type proteins, or the phosphorylated T527A/T163A mutant proteins A) elute at a later volume than the phosphorylated wild type proteins and B) bind to a lower proportion of the MBP-PLK1PBD (as you can see in the relative absorbance profiles and Coomassie gels).

   • The authors would need to provide convincing evidence that co-depletion of BUB-1 and HCP-4 delocalizes PLK-1 from the chromosomes entirely, and that this co-depletion condition is more severe than either single depletion alone.

   We now provide a quantitation on the total PLK-1 levels to go along the images (New Figure 8-figure supplement 1).

   Additionally, the bub-1T527A and hcp-4T163A alleles are nice tools to, in theory, more specifically delocalize PLK-1 from the midbivalent and chromosome arms, respectively, to explore the functions of chromosome-associated PLK-1. However, I think the authors cannot rule out the possibility that other proteins are also being depleted from the midbivalent and/or chromosome arms in their conditions, and that this delocalization may contribute to the phenotypes observed. For example, hcp-4 depletion was recently shown to delocalize KLP-19 from the chromosome arms (Horton et.al. 2022), so in the experiment shown in Figure 6E (HCP-4 RNAi in the bub-1 mutant), PLK-1 was likely not the only protein missing from the chromosome arms. Therefore, understanding if other proteins are absent from these domains (in the bub-1T527A and hcp-4T16A3 mutants) would help the reader understand and interpret the presented phenotypes (and how specific they are to PLK-1 loss). Consequently, I think that to better understand the co-depletion analysis presented in Figure 6 (and Figure 6 supplement 1), the authors should analyze other midbivalent and chromosome arm proteins, to determine if any are also delocalized (e.g. SUMO, KLP-19, MCAK, etc.).

   As stated above, this paper focuses on identifying the specific meiotic events PLK-1 plays a role in and characterising its targeting mechanism. We are following on this work to understand what proteins are regulated by PLK-1 in different chromosome domains and how this relates to the observed phenotypes.

   For the current, we should emphasise that mutating a single Thr residue within an STP motif in a largely disordered region is far more specific than depleting HCP-4 or BUB-1, making it likely that the observed effects are mediated through PLK-1 targeting. It should be noted that the finding presented in Horton et.al. 2022 is in contradiction with another study in which hcp-4 depletion did not impact KLP-19 localisation (Hattersley et al 2022).

   Additionally, instead of performing a combination of mutant and RNAi analysis (i.e. HCP-4 RNAi in the bub-1 mutant (Figure 6) and BUB-1 RNAi in the hcp-4 mutant (Figure 6 figure supplement 1)), it would be more powerful to generate a double mutant - this has a higher chance of being a more specific depletion condition.

   We have performed these experiments, which are now presented in Figure 9.

   Read the original source
3. 

   Version published to 10.1101/2022.10.07.511262v2 on bioRxiv

   Mar 28, 2023
4. 

   eLife

   Dec 7, 2022

   eLife assessment

   This fundamental work addresses the role of the mitotic kinase PLK-1 in meiosis, using C. elegans as a model system. The valuable findings are convincing and combine beautiful cell biology and biochemical assays. The work will be of broad interest to people working on Plk1 and/or in meiosis in many different systems.

   Read the original source
5. 

   eLife

   Dec 7, 2022

   Reviewer #1 (Public Review):

   This study focuses on the role of polo like kinase 1 (PLK-1) during oocyte meiosis. In mammalian oocytes, Plk1 localizes to chromosomes and spindle poles, and there is evidence that it is required for nuclear envelope breakdown, spindle formation, chromosome segregation, and polar body extrusion. However, how Plk1 is targeted to its various locations and how it performs these functions is not well understood. This study uses C. elegans oocytes as a model to explore PLK-1 function during meiosis. They take advantage of an analogue-sensitive allele of plk-1, which enabled them to bypass nuclear envelope breakdown defects that occur following PLK-1 RNAi. This allowed them to dissect later roles of PLK-1 in oocytes, demonstrating that depletion causes defects in spindle organization, chromosome congression, …

   Reviewer #1 (Public Review):

   This study focuses on the role of polo like kinase 1 (PLK-1) during oocyte meiosis. In mammalian oocytes, Plk1 localizes to chromosomes and spindle poles, and there is evidence that it is required for nuclear envelope breakdown, spindle formation, chromosome segregation, and polar body extrusion. However, how Plk1 is targeted to its various locations and how it performs these functions is not well understood. This study uses C. elegans oocytes as a model to explore PLK-1 function during meiosis. They take advantage of an analogue-sensitive allele of plk-1, which enabled them to bypass nuclear envelope breakdown defects that occur following PLK-1 RNAi. This allowed them to dissect later roles of PLK-1 in oocytes, demonstrating that depletion causes defects in spindle organization, chromosome congression, segregation, and polar body extrusion. Moreover, the authors defined mechanisms by which PLK-1 is targeted to chromosomes, showing that CENP-C (HCP-4) is required for localization to chromosome arms and that BUB-1 is required for targeting to the midbivalent region. Finally, they demonstrate that upon removal of PLK-1 from both domains, there are severe meiotic defects. These findings are interesting. However, there is a need for additional analysis to better support some of their conclusions, and to aid in interpretation of particular phenotypes. Specific comments are below.

   - For many important claims of the paper, a single representative image is shown but the n is not noted. This is an issue throughout the paper for much of the localization analysis (e.g. Figure 1B, 1C, 1D, 2A, 2B, 3A, 3B, 3C, etc.); in cases like this, numbers should be included to increase the rigor of the presented data. How many images or movies were analyzed that looked like the one shown? For linescans, were they done only on one image? How many independent experiments were done, etc?.

   - In the abstract, it is stated that PLK-1 plays a role in spindle assembly/stability (this is also stated elsewhere, e.g. line 101). This phrasing implies that the authors have demonstrated roles in both spindle assembly and stability. However, to distinguish between these roles, they would have to show that removal of PLK-1 before spindle assembly causes defects, and also that removal of PLK-1 from pre-formed spindles causes collapse. I don't think it is necessary to do this, as the spindle roles of PLK-1 are not a focus of the paper. However, the language should be altered so that it does not imply that the paper has demonstrated roles in both. A good place to do this would be in the section from lines 144-147, where they first discuss the spindle defects. It would be straightforward to explain that their approach does not distinguish between spindle assembly and stability, and that PLK-1 could have a role in either or both.

   - It is stated that there is kinetochore localization of PLK-1 (and I do see some dim cup-like localization in images after PLK-1 is removed from the chromosome arms via HCP-4 RNAi). However, this cup-like localization is not clear in most wild-type images (e.g. Figure 1B, 1D, 2A, 3A, etc.). Although I recognize that the chromatin staining might be obscuring kinetochore localization, if PLK-1 was truly a kinetochore protein I would also expect it to localize to filaments within the spindle (as many other kinetochore proteins do), especially since the authors state that BUB-1 targets PLK-1 to the kinetochore (and BUB-1 is in the filaments). In fact, the only images where it looks like PLK-1 may be localized to filaments are in Figure 4C and 6A, when HCP-4 has been depleted (though I don't know if this generally true across all HCP-4 RNAi images). For me, this calls into question the conclusion that PLK-1 truly is on the kinetochore in wild type conditions - could it be that PLK-1 only localizes to the kinetochore (and to the filaments) when HCP-4 is depleted? The authors need to resolve this issue and provide better evidence that PLK-1 normally localizes to the kinetochore, if they want to make this claim. Additionally, the observation that PLK-1 is not on the kinetochore filaments (in wild type conditions) should be addressed in the text somewhere - do the authors think that this is a special type of kinetochore protein that does not localize to the filaments?

   - The authors should provide a control experiment, treating wild-type worms with 10uM 3-IB-PP1. This would be important to ensure that the spindle defects seen at this concentration in the plk-1as strain are not non-specific effects of the inhibitor. There is a control in Figure 1 - figure supplement 3 using 1uM 3-IB-PP1 but didn't see a control for 10uM (the concentration at which spindle defects are observed).

   - In Figure 2F, the gels for BUB-1+PLK-1 look different in the presence and absence of phosphorylation by Cdk1 - for these data, I agree with the authors that it looks as if the complex elutes at a higher volume if BUB-1 is not phosphorylated (lines 200-204). However, Figure 2G has a repeat of the condition with phosphorylated BUB-1, and in this panel, the complex appears to elute at a higher volume than it did on the gel in panel F. The gel in panel G looks much more similar to the unphosphorylated condition in panel F. The authors need to explain this discrepancy (i.e., Is there a reason why the gels cannot be compared between panels? How reproducible are these data?). Ideally, the authors would include a repeat of the unphosphorylated BUB-1 + PLK-1 condition in panel G, done at the same time as the conditions shown in that panel, to avoid the impression that their results may not be reproducible.

   - The authors would need to provide convincing evidence that co-depletion of BUB-1 and HCP-4 delocalizes PLK-1 from the chromosomes entirely, and that this co-depletion condition is more severe than either single depletion alone. Additionally, the bub-1T527A and hcp-4T163A alleles are nice tools to, in theory, more specifically delocalize PLK-1 from the midbivalent and chromosome arms, respectively, to explore the functions of chromosome-associated PLK-1. However, I think the authors cannot rule out the possibility that other proteins are also being depleted from the midbivalent and/or chromosome arms in their conditions, and that this delocalization may contribute to the phenotypes observed. For example, hcp-4 depletion was recently shown to delocalize KLP-19 from the chromosome arms (Horton et.al. 2022), so in the experiment shown in Figure 6E (HCP-4 RNAi in the bub-1 mutant), PLK-1 was likely not the only protein missing from the chromosome arms. Therefore, understanding if other proteins are absent from these domains (in the bub-1T527A and hcp-4T16A3 mutants) would help the reader understand and interpret the presented phenotypes (and how specific they are to PLK-1 loss). Consequently, I think that to better understand the co-depletion analysis presented in Figure 6 (and Figure 6 supplement 1), the authors should analyze other midbivalent and chromosome arm proteins, to determine if any are also delocalized (e.g. SUMO, KLP-19, MCAK, etc.). Additionally, instead of performing a combination of mutant and RNAi analysis (i.e. HCP-4 RNAi in the bub-1 mutant (Figure 6) and BUB-1 RNAi in the hcp-4 mutant (Figure 6 figure supplement 1)), it would be more powerful to generate a double mutant - this has a higher chance of being a more specific depletion condition.

   Read the original source
6. 

   eLife

   Dec 7, 2022

   Reviewer #2 (Public Review):

   In this manuscript, Taylor et al. analyzed the role of the Polo-like kinase PLK-1 during female meiosis in the C. elegans oocyte. By temporally inhibiting an analogue-sensitive PLK-1 mutant (bypassing the PLK-1 requirement for nuclear envelope breakdown) they demonstrate that PLK-1 is involved in meiotic spindle assembly and/or stability, chromosome alignment and polar body extrusion. Consistent with its role in these processes, the authors demonstrate that PLK-1 localizes to multiple regions of the meiotic spindle: the spindle poles, chromosome arms, kinetochores and midbivalent region between the homologous chromosomes during meiosis I. They further dissected the mechanism recruiting PLK-1 to these structures and showed that CENP-CHCP-4 recruits PLK-1 to the chromosome arms while BUB-1 recruits PLK-1 to …

   Reviewer #2 (Public Review):

   In this manuscript, Taylor et al. analyzed the role of the Polo-like kinase PLK-1 during female meiosis in the C. elegans oocyte. By temporally inhibiting an analogue-sensitive PLK-1 mutant (bypassing the PLK-1 requirement for nuclear envelope breakdown) they demonstrate that PLK-1 is involved in meiotic spindle assembly and/or stability, chromosome alignment and polar body extrusion. Consistent with its role in these processes, the authors demonstrate that PLK-1 localizes to multiple regions of the meiotic spindle: the spindle poles, chromosome arms, kinetochores and midbivalent region between the homologous chromosomes during meiosis I. They further dissected the mechanism recruiting PLK-1 to these structures and showed that CENP-CHCP-4 recruits PLK-1 to the chromosome arms while BUB-1 recruits PLK-1 to the midbivalent and kinetochores. The interaction between PLK-1 and its partners is mediated by phosphorylation of a Polo-docking site (consensus STP) in BUB-1 and CENP-CHCP-4. Finally, the authors show that both PLK-1 recruitment pathways are critically required for PLK-1 function in female meiosis.

   This fundamental work substantially advances our understanding of PLK-1 function during female meiosis.
   Overall, the data presented are of very high quality and support the major conclusions of the paper with one or two exceptions.

   Read the original source
7. 

   eLife

   Dec 7, 2022

   Reviewer #3 (Public Review):

   This is a very well written manuscript which addresses the role of the mitotic kinase Polo like kinase 1 in meiosis using the C. elegans fertilized oocyite as a model system. The authors show that PLK-1 localizes at different locations on meiotic spindles and chromosomes and identify the mechanisms required for the different localization patterns. Finally, the authors show which pool of PLK-1 is required for the different functions of PLK-1 in meiosis, using the power of genetics via CRISPR.

   The strengths of the manuscript are the temporal inhibition of PLK-1 to study the meiotic roles of this kinase, the identification of the mechanisms that control PLK-1 localization and how this is regulated (phosphorylation) and the combination of cell biology and biochemstry.

   This work will be of high interest to both …

   Reviewer #3 (Public Review):

   This is a very well written manuscript which addresses the role of the mitotic kinase Polo like kinase 1 in meiosis using the C. elegans fertilized oocyite as a model system. The authors show that PLK-1 localizes at different locations on meiotic spindles and chromosomes and identify the mechanisms required for the different localization patterns. Finally, the authors show which pool of PLK-1 is required for the different functions of PLK-1 in meiosis, using the power of genetics via CRISPR.

   The strengths of the manuscript are the temporal inhibition of PLK-1 to study the meiotic roles of this kinase, the identification of the mechanisms that control PLK-1 localization and how this is regulated (phosphorylation) and the combination of cell biology and biochemstry.

   This work will be of high interest to both the Polo like kinase and the meiotic communities.

   Read the original source
8. 

   Version published to 10.1101/2022.10.07.511262v1 on bioRxiv

   Oct 7, 2022