Synergistic stabilization of microtubules by BUB-1, HCP-1, and CLS-2 controls microtubule pausing and meiotic spindle assembly
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eLife assessment
This paper on the regulation of microtubule dynamics during C. elegans meiosis will be of interest to scientists in the broad field of microtubule function in both mitosis and meiosis. The experiments are beautifully conducted and presented and generally support the conclusions of the paper. The results are interesting and add to our understanding of the control of microtubule dynamics at the kinetochore and its functional consequences for meiosis.
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Abstract
During cell division, chromosome segregation is orchestrated by a microtubule-based spindle. Interaction between spindle microtubules and kinetochores is central to the bi-orientation of chromosomes. Initially dynamic to allow spindle assembly and kinetochore attachments, which is essential for chromosome alignment, microtubules are eventually stabilized for efficient segregation of sister chromatids and homologous chromosomes during mitosis and meiosis I, respectively. Therefore, the precise control of microtubule dynamics is of utmost importance during mitosis and meiosis. Here, we study the assembly and role of a kinetochore module, comprised of the kinase BUB-1, the two redundant CENP-F orthologs HCP-1/2, and the CLASP family member CLS-2 (hereafter termed the BHC module), in the control of microtubule dynamics in Caenorhabditis elegans oocytes. Using a combination of in vivo structure-function analyses of BHC components and in vitro microtubule-based assays, we show that BHC components stabilize microtubules, which is essential for meiotic spindle formation and accurate chromosome segregation. Overall, our results show that BUB-1 and HCP-1/2 do not only act as targeting components for CLS-2 at kinetochores, but also synergistically control kinetochore-microtubule dynamics by promoting microtubule pause. Together, our results suggest that BUB-1 and HCP-1/2 actively participate in the control of kinetochore-microtubule dynamics in the context of an intact BHC module to promote spindle assembly and accurate chromosome segregation in meiosis.
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eLife assessment
This paper on the regulation of microtubule dynamics during C. elegans meiosis will be of interest to scientists in the broad field of microtubule function in both mitosis and meiosis. The experiments are beautifully conducted and presented and generally support the conclusions of the paper. The results are interesting and add to our understanding of the control of microtubule dynamics at the kinetochore and its functional consequences for meiosis.
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Reviewer #1 (Public Review):
This manuscript investigates the assembly and function of BUB-1, HCP-1/2 (CenpF) and CLS-2 (CLASP), which they call the BHC module, at the kinetochores. The experiments were executed at a high standard. The data is clearly presented and generally support the conclusions.
Independency of Bub-1 kinetochore localization from Bub-3 is novel and different from humans or yeast. Detailed analysis of interaction domains among the BHC module is well carried out. Showing the redundant roles of the kinetochore and ring localizations of CLS-2 is an interesting result. They further present in vivo and in vitro evidences that Bub-1 and HCP-1/2 are not simply recruiting CLS-2 to the kinetochores, but also enhance the activity of CLS-2. These findings provide a significant insight into how the BHC module functions together. …
Reviewer #1 (Public Review):
This manuscript investigates the assembly and function of BUB-1, HCP-1/2 (CenpF) and CLS-2 (CLASP), which they call the BHC module, at the kinetochores. The experiments were executed at a high standard. The data is clearly presented and generally support the conclusions.
Independency of Bub-1 kinetochore localization from Bub-3 is novel and different from humans or yeast. Detailed analysis of interaction domains among the BHC module is well carried out. Showing the redundant roles of the kinetochore and ring localizations of CLS-2 is an interesting result. They further present in vivo and in vitro evidences that Bub-1 and HCP-1/2 are not simply recruiting CLS-2 to the kinetochores, but also enhance the activity of CLS-2. These findings provide a significant insight into how the BHC module functions together. Why the kinetochore and ring localizations of CLS-2 are redundant and how regulation of single microtubules is linked to the overall spindle assembly have not been addressed, but I think that they are beyond the scope of this manuscript.
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Reviewer #2 (Public Review):
Macaisne and colleagues investigate the assembly and function of a protein module consisting of the kinase BUB-1 and the microtubule binding proteins HCP-1/CENP-F and CLS-2/CLASP, which function at kinetochores during cell division. By replacing endogenous proteins with RNAi-resistant transgenic mutants that are expressed at endogenous levels, the authors screen for protein domains involved in recruitment of the module to meiotic kinetochores in oocytes. This tour de force clarifies the connectivity among the components of the module and confirms a linear assembly hierarchy in which the outer kinetochore protein KNL-1 recruits BUB-1 (surprisingly independently of its binding partner BUB-3), BUB-1 recruits HCP-1, and HCP-1 recruits CLS-2. Having identified deletion mutants that perturb specific interactions …
Reviewer #2 (Public Review):
Macaisne and colleagues investigate the assembly and function of a protein module consisting of the kinase BUB-1 and the microtubule binding proteins HCP-1/CENP-F and CLS-2/CLASP, which function at kinetochores during cell division. By replacing endogenous proteins with RNAi-resistant transgenic mutants that are expressed at endogenous levels, the authors screen for protein domains involved in recruitment of the module to meiotic kinetochores in oocytes. This tour de force clarifies the connectivity among the components of the module and confirms a linear assembly hierarchy in which the outer kinetochore protein KNL-1 recruits BUB-1 (surprisingly independently of its binding partner BUB-3), BUB-1 recruits HCP-1, and HCP-1 recruits CLS-2. Having identified deletion mutants that perturb specific interactions among module components, the authors use these separation-of-function mutants to investigate how the module contributes to female meiotic divisions using live cell imaging. The results allow the authors to conclude that the module has both kinetochore-dependent and kinetochore-independent functions and that module integrity is important for spindle assembly and chromosome segregation. In an elegant domain-swapping experiment the authors target CLS-2 directly to BUB-1 so that HCP-1 is no longer necessary for CLS-2 recruitment. Depletion of HCP-1 in this background reveals that HCP-1's role goes beyond that of a CLS-2 recruitment factor. Finally, an in-depth mutational analysis of CLS-2's microtubule binding region shows that only one of the two TOG-like domains is essential for CLS-2 function, consistent with the absence of critical residues in the second TOG-like domain. The extensive in vivo analysis of module mutants is complemented by in vitro assays that directly assess the effect of module components on microtubule dynamics. This confirms CLS-2's role as a microtubule stabilizer but also reveals that addition of the other two components modulates this effect.
The experiments presented in this paper are rigorous and succeed in elucidating the functional relevance of the interactions among BUB-1, HCP-1, and CLS-2. The main conclusion of the paper, namely that these components work as a unit, is well supported by the in vivo evidence. What is less clear is whether the effects observed in vitro reflect the activity of the intact module. This part of the paper would profit from analysis of binding-defective mutants. Specifically, including HCP-1 mutants defective in CLS-2 and/or BUB-1 binding would help determine whether the enhancement of microtubule pausing that is observed in the presence of all three components requires assembly of the module.
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Reviewer #3 (Public Review):
Macaisne et al., use C. elegans oocytes to investigate the function of the kinetochore localised BHC module composed of BUB-1 (homologue of mammalian Bub1), HCP1/2 (homologue of mammalian CENP-F) and CLS-2 (homologue of mammalian CLASP) in meiotic spindle regulation. Since defects in meiotic spindle assembly would lead to defective meiotic chromosome segregation, known to give rise to birth defects, this is an important area of research. In the first part of the paper, the authors determine the domains of the BHC module and outer kinetochore components that are involved in localising the complex to kinetochores or ring domains of meiotic bivalent chromosomes. The functional consequences of BHC module mis-localisation are then assessed by live cell imaging. The authors find that a correctly assembled BHC …
Reviewer #3 (Public Review):
Macaisne et al., use C. elegans oocytes to investigate the function of the kinetochore localised BHC module composed of BUB-1 (homologue of mammalian Bub1), HCP1/2 (homologue of mammalian CENP-F) and CLS-2 (homologue of mammalian CLASP) in meiotic spindle regulation. Since defects in meiotic spindle assembly would lead to defective meiotic chromosome segregation, known to give rise to birth defects, this is an important area of research. In the first part of the paper, the authors determine the domains of the BHC module and outer kinetochore components that are involved in localising the complex to kinetochores or ring domains of meiotic bivalent chromosomes. The functional consequences of BHC module mis-localisation are then assessed by live cell imaging. The authors find that a correctly assembled BHC module is indispensable for correct chromosome segregation during meiosis. Using recombinantly expressed proteins, the authors then show that in vitro the components of the BHC module synergistically regulate microtubule behaviour. In particular, the incidence of pausing during microtubule growth was significantly increased by the addition of all three BHC components. This is interesting because BUB-1 by itself did not influence microtubule growth properties hence only seems to exert its influence in a complex with HCP1/2 and CLS-2.
Strengths:
The data presented in the manuscript are generally of very high quality and very nicely presented, and the effects observed are convincing and confirm the statements in the manuscript text. The analysis of the purified proteins of interest in an in vitro setting adds an extra dimension to the study and is highly informative since it shows that the combined actions of the BHC proteins results in the strong promotion of microtubule growth pausing.
Weaknesses:
While the combination of live cell imaging and in vitro essays with purified proteins is one of the strengths of the manuscripts, it also highlights a gap in the understanding of the function of the BHC module. How does the ability of this complex to induce pausing in microtubule growth relate to the observed defects in chromosome segregation in oocytes expressing defective BHC components? What are the precise molecular deficiencies causing the mis-segregation? Could the authors investigate this more directly than by just measuring spindle microtubule density? Is the spindle assembly checkpoint activated by the BHC module modifications that the authors test? Some of the conditions seem to result in delayed timing of meiosis consistent with this idea.
Although the analysis of the process of meiosis in C. elegans oocytes has interesting implications for mitosis and meiosis in other systems, it is a very specialised system, that not all readers may be entirely familiar with. A more extensive discussion, comparing systems and highlighting points of diversion would therefore be useful for many readers.
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