Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts

  1. Charlotte Guyomar
  2. Clément Bousquet
  3. Siou Ku
  4. John M Heumann
  5. Gabriel Guilloux
  6. Natacha Gaillard
  7. Claire Heichette
  8. Laurence Duchesne
  9. Michel O Steinmetz
  10. Romain Gibeaux
  11. Denis Chrétien  Is a corresponding author
  1. Université de Rennes 1, CNRS, France
  2. University of Colorado Boulder, United States
  3. Paul Scherrer Institute, Switzerland

Abstract

Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions including motility, compartmentalization, and division. They are considered as pseudo-helical polymers whose constituent ab-tubulin heterodimers share lateral homotypic interactions, except at one unique region called the seam. Here, we used a segmented sub-tomogram averaging strategy to reassess this paradigm and analyze the organization of the ab-tubulin heterodimers in microtubules assembled from purified porcine brain tubulin in the presence of GTP and GMPCPP, and in Xenopus egg cytoplasmic extracts. We find that in almost all conditions, microtubules incorporate variable protofilament and/or tubulin subunit helical-start numbers, as well as variable numbers of seams. Strikingly, the seam number and location vary along individual microtubules, generating holes of one to a few subunits in size within their lattices. Together, our results reveal that the formation of mixed and discontinuous microtubule lattices is an intrinsic property of tubulin that requires the formation of unique lateral interactions without longitudinal ones. They further suggest that microtubule assembly is tightly regulated in a cytoplasmic environment.

Data availability

Sub-tomogram averages and extracts from cryo-electron tomograms presented in the figures have been deposited onto the EMDB and are listed in Supplementary Table 1 with reference to the corresponding figures and videos. All the tilt series, tomograms, models and motiv lists used to reconstruct the microtubule segments in PEET have been deposited onto the EMPIAR (Supplementary Table 2).

The following data sets were generated

Article and author information

Author details

  1. Charlotte Guyomar

    Institut de Génétique et Développement de Rennes, Université de Rennes 1, CNRS, Rennes, France
    Competing interests
    The authors declare that no competing interests exist.
  2. Clément Bousquet

    Institut de Génétique et Développement de Rennes, Université de Rennes 1, CNRS, Rennes, France
    Competing interests
    The authors declare that no competing interests exist.
  3. Siou Ku

    Institut de Génétique et Développement de Rennes, Université de Rennes 1, CNRS, Rennes, France
    Competing interests
    The authors declare that no competing interests exist.
  4. John M Heumann

    Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Gabriel Guilloux

    Institut de Génétique et Développement de Rennes, Université de Rennes 1, CNRS, Rennes, France
    Competing interests
    The authors declare that no competing interests exist.
  6. Natacha Gaillard

    Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  7. Claire Heichette

    Institut de Génétique et Développement de Rennes, Université de Rennes 1, CNRS, Rennes, France
    Competing interests
    The authors declare that no competing interests exist.
  8. Laurence Duchesne

    Institut de Génétique et Développement de Rennes, Université de Rennes 1, CNRS, Rennes, France
    Competing interests
    The authors declare that no competing interests exist.
  9. Michel O Steinmetz

    Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  10. Romain Gibeaux

    Institut de Génétique et Développement de Rennes, Université de Rennes 1, CNRS, Rennes, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5081-1985
  11. Denis Chrétien

    Institute of Genetics and Development of Rennes, Université de Rennes 1, CNRS, Rennes, France
    For correspondence
    denis.chretien@univ-rennes1.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8261-4396

Funding

Agence Nationale de la Recherche (ANR-16-CE11-0017-01)

  • Denis Chrétien

Agence Nationale de la Recherche (ANR-18-CE13-0001-01)

  • Denis Chrétien

Human Frontier Science Program (CDA00019/1019-C)

  • Romain Gibeaux

Swiss National Science Fondation (310030_192566)

  • Michel O Steinmetz

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

Ethics

Animal experimentation: All animal experimentation in this study was performed according to our animal use protocol APAFiS #26858-2020072110205978 approved by the Animal Use Ethic Committee (#7, Rennes, France) and the French Ministry of Higher Education, Research and Innovation. Mature Xenopus laevis female frogs were obtained from the CRB Xénope (Rennes, France) and ovulated with no harm to the animals with at least a 6-month rest interval between ovulations.

Copyright

© 2022, Guyomar 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

  • 2,536
    views
  • 265
    downloads
  • 12
    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. Charlotte Guyomar
  2. Clément Bousquet
  3. Siou Ku
  4. John M Heumann
  5. Gabriel Guilloux
  6. Natacha Gaillard
  7. Claire Heichette
  8. Laurence Duchesne
  9. Michel O Steinmetz
  10. Romain Gibeaux
  11. Denis Chrétien
(2022)
Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts
eLife 11:e83021.
https://doi.org/10.7554/eLife.83021

Share this article

https://doi.org/10.7554/eLife.83021

Further reading

    1. Cell Biology
    2. Chromosomes and Gene Expression
    Bhumil Patel, Maryke Grobler ... Needhi Bhalla
    Research Article

    Meiotic crossover recombination is essential for both accurate chromosome segregation and the generation of new haplotypes for natural selection to act upon. This requirement is known as crossover assurance and is one example of crossover control. While the conserved role of the ATPase, PCH-2, during meiotic prophase has been enigmatic, a universal phenotype when pch-2 or its orthologs are mutated is a change in the number and distribution of meiotic crossovers. Here, we show that PCH-2 controls the number and distribution of crossovers by antagonizing their formation. This antagonism produces different effects at different stages of meiotic prophase: early in meiotic prophase, PCH-2 prevents double-strand breaks from becoming crossover-eligible intermediates, limiting crossover formation at sites of initial double-strand break formation and homolog interactions. Later in meiotic prophase, PCH-2 winnows the number of crossover-eligible intermediates, contributing to the designation of crossovers and ultimately, crossover assurance. We also demonstrate that PCH-2 accomplishes this regulation through the meiotic HORMAD, HIM-3. Our data strongly support a model in which PCH-2’s conserved role is to remodel meiotic HORMADs throughout meiotic prophase to destabilize crossover-eligible precursors and coordinate meiotic recombination with synapsis, ensuring the progressive implementation of meiotic recombination and explaining its function in the pachytene checkpoint and crossover control.

    1. Cell Biology
    2. Physics of Living Systems
    Marta Urbanska, Yan Ge ... Jochen Guck
    Research Article

    Cell mechanical properties determine many physiological functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors that govern the mechanical properties is therefore a subject of great interest. Here, we present a mechanomics approach for establishing links between single-cell mechanical phenotype changes and the genes involved in driving them. We combine mechanical characterization of cells across a variety of mouse and human systems with machine learning-based discriminative network analysis of associated transcriptomic profiles to infer a conserved network module of five genes with putative roles in cell mechanics regulation. We validate in silico that the identified gene markers are universal, trustworthy, and specific to the mechanical phenotype across the studied mouse and human systems, and demonstrate experimentally that a selected target, CAV1, changes the mechanical phenotype of cells accordingly when silenced or overexpressed. Our data-driven approach paves the way toward engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions.