Synergistic stabilization of microtubules by BUB-1, HCP-1 and CLS-2 controls microtubule pausing and meiotic spindle assembly

  1. Nicolas Macaisne
  2. Laura Bellutti
  3. Kimberley Laband
  4. Frances Edwards
  5. Laras Pitayu-Nugroho
  6. Alison Gervais
  7. Thadshagine Ganeswaran
  8. Hélène Geoffroy
  9. Gilliane Maton
  10. Julie C Canman
  11. Benjamin Lacroix  Is a corresponding author
  12. Julien Dumont  Is a corresponding author
  1. Institut Jacques Monod, France
  2. Columbia University, United States
  3. Centre de Recherche en Biologie Cellulaire de Montpellier, France

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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files; Source Data files have been provided for all figures.

Article and author information

Author details

  1. Nicolas Macaisne

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0109-9845
  2. Laura Bellutti

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  3. Kimberley Laband

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8535-2050
  4. Frances Edwards

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  5. Laras Pitayu-Nugroho

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  6. Alison Gervais

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  7. Thadshagine Ganeswaran

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4986-4419
  8. Hélène Geoffroy

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3231-8369
  9. Gilliane Maton

    Institut Jacques Monod, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  10. Julie C Canman

    Department of Pathology and Cell, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Benjamin Lacroix

    Centre de Recherche en Biologie Cellulaire de Montpellier, Montpellier, France
    For correspondence
    benjamin.lacroix@crbm.cnrs.fr
    Competing interests
    The authors declare that no competing interests exist.
  12. Julien Dumont

    Institut Jacques Monod, Paris, France
    For correspondence
    julien.dumont@ijm.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5312-9770

Funding

Agence Nationale de la Recherche (ANR-19-CE13-0015)

  • Nicolas Macaisne

Fondation pour la Recherche Médicale (Post Doctoral Fellowship)

  • Laura Bellutti

European Research Council (CoG Chromosome 819179)

  • Julien Dumont

Agence Nationale de la Recherche (ANR-19-CE13-0015)

  • Julien Dumont

National Institutes of Health (R01GM117407)

  • Julie C Canman

National Institutes of Health (R01GM130764)

  • Julie C Canman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2023, Macaisne et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,227
    views
  • 186
    downloads
  • 9
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Nicolas Macaisne
  2. Laura Bellutti
  3. Kimberley Laband
  4. Frances Edwards
  5. Laras Pitayu-Nugroho
  6. Alison Gervais
  7. Thadshagine Ganeswaran
  8. Hélène Geoffroy
  9. Gilliane Maton
  10. Julie C Canman
  11. Benjamin Lacroix
  12. Julien Dumont
(2023)
Synergistic stabilization of microtubules by BUB-1, HCP-1 and CLS-2 controls microtubule pausing and meiotic spindle assembly
eLife 12:e82579.
https://doi.org/10.7554/eLife.82579

Share this article

https://doi.org/10.7554/eLife.82579

Further reading

    1. Cell Biology
    2. Genetics and Genomics
    Keva Li, Nicholas Tolman ... UK Biobank Eye and Vision Consortium
    Research Article

    A glaucoma polygenic risk score (PRS) can effectively identify disease risk, but some individuals with high PRS do not develop glaucoma. Factors contributing to this resilience remain unclear. Using 4,658 glaucoma cases and 113,040 controls in a cross-sectional study of the UK Biobank, we investigated whether plasma metabolites enhanced glaucoma prediction and if a metabolomic signature of resilience in high-genetic-risk individuals existed. Logistic regression models incorporating 168 NMR-based metabolites into PRS-based glaucoma assessments were developed, with multiple comparison corrections applied. While metabolites weakly predicted glaucoma (Area Under the Curve = 0.579), they offered marginal prediction improvement in PRS-only-based models (p=0.004). We identified a metabolomic signature associated with resilience in the top glaucoma PRS decile, with elevated glycolysis-related metabolites—lactate (p=8.8E-12), pyruvate (p=1.9E-10), and citrate (p=0.02)—linked to reduced glaucoma prevalence. These metabolites combined significantly modified the PRS-glaucoma relationship (Pinteraction = 0.011). Higher total resilience metabolite levels within the highest PRS quartile corresponded to lower glaucoma prevalence (Odds Ratiohighest vs. lowest total resilience metabolite quartile=0.71, 95% Confidence Interval = 0.64–0.80). As pyruvate is a foundational metabolite linking glycolysis to tricarboxylic acid cycle metabolism and ATP generation, we pursued experimental validation for this putative resilience biomarker in a human-relevant Mus musculus glaucoma model. Dietary pyruvate mitigated elevated intraocular pressure (p=0.002) and optic nerve damage (p<0.0003) in Lmx1bV265D mice. These findings highlight the protective role of pyruvate-related metabolism against glaucoma and suggest potential avenues for therapeutic intervention.

    1. Cell Biology
    Affiong Ika Oqua, Kin Chao ... Alejandra Tomas
    Research Article

    G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics, and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering, and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active vs inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these, Val229, to alanine on GLP-1R-cholesterol interactions, plasma membrane behaviours, clustering, trafficking and signalling in INS-1 832/3 rat pancreatic beta cells and primary mouse islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study (1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; (2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; (3) validates their functional relevance in beta cells; and (4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.