A nanobody toolbox to investigate localisation and dynamics of Drosophila titins and other key sarcomeric proteins

  1. Vincent Loreau
  2. Renate Rees
  3. Eunice HoYee Chan
  4. Waltraud Taxer
  5. Kathrin Gregor
  6. Bianka Mußil
  7. Christophe Pitaval
  8. Nuno Miguel Luis
  9. Pierre Mangeol
  10. Frank Schnorrer  Is a corresponding author
  11. Dirk Görlich  Is a corresponding author
  1. Aix Marseille University, CNRS, IDBM, France
  2. Max Planck Institute for Multidisciplinary Sciences, Germany

Abstract

Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understanding protein function. However, to date, this is challenging, as the accessibility of large antibodies to dense tissues is often limited, and fluorescent proteins inserted close to a domain of interest may affect protein function. These complications apply in particular to muscle sarcomeres, arguably one of the most protein-dense assemblies in nature, which complicates studying sarcomere morphogenesis at molecular resolution. Here, we introduce a toolbox of nanobodies recognising various domains of the two Drosophila titin homologs, Sallimus and Projectin, as well as the key sarcomeric proteins Obscurin, a-Actinin and Zasp52. We verified the superior labelling qualities of our nanobodies in muscle tissue as compared to antibodies. By applying our toolbox to larval muscles, we found a gigantic Sallimus isoform stretching more than 2 µm to bridge the sarcomeric I-band, while Projectin covers almost the entire myosin filaments in a polar orientation. Transgenic expression of tagged nanobodies confirmed their high affinity-binding without affecting target protein function. Finally, adding a degradation signal to anti-Sallimus nanobodies suggested that it is difficult to fully degrade Sallimus in mature sarcomeres, however expression of these nanobodies caused developmental lethality. These results may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

Data availability

All quantitative source data are provided. Newly generated code is publicly available here: https://github.com/PierreMangeol/titin_PAINTE.coli nanobody expression vectors are available from Addgene (https://www.addgene.org/depositing/82080/).

Article and author information

Author details

  1. Vincent Loreau

    Turing Centre for Living Systems, Aix Marseille University, CNRS, IDBM, Marseille, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0556-2825
  2. Renate Rees

    Department of Cellular Logistics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Eunice HoYee Chan

    Turing Centre for Living Systems, Aix Marseille University, CNRS, IDBM, Marseille, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3162-3609
  4. Waltraud Taxer

    Department of Cellular Logistics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Kathrin Gregor

    Department of Cellular Logistics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Bianka Mußil

    Department of Cellular Logistics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Christophe Pitaval

    Turing Centre for Living Systems, Aix Marseille University, CNRS, IDBM, Marseille, France
    Competing interests
    The authors declare that no competing interests exist.
  8. Nuno Miguel Luis

    Turing Centre for Living Systems, Aix Marseille University, CNRS, IDBM, Marseille, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5438-9638
  9. Pierre Mangeol

    Turing Centre for Living Systems, Aix Marseille University, CNRS, IDBM, Marseille, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8305-7322
  10. Frank Schnorrer

    Turing Centre for Living Systems, Aix Marseille University, CNRS, IDBM, Marseille, France
    For correspondence
    frank.schnorrer@univ-amu.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9518-7263
  11. Dirk Görlich

    Department of Cellular Logistics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
    For correspondence
    goerlich@mpinat.mpg.de
    Competing interests
    The authors declare that no competing interests exist.

Funding

Centre National de la Recherche Scientifique

  • Frank Schnorrer

Agence Nationale de la Recherche (ANR-ACHN MUSCLE-FORCES)

  • Frank Schnorrer

Human Frontier Science Program (RGP0052/2018)

  • Frank Schnorrer

Bettencourt Schueller Foundation

  • Frank Schnorrer

Agence Nationale de la Recherche (ANR-10-INBS-04-01)

  • Frank Schnorrer

Agence Nationale de la Recherche (ANR-16-CONV-0001)

  • Frank Schnorrer

Aix-Marseille Université (Center for Living Systems)

  • Frank Schnorrer

Aix-Marseille Université (LabEx-INFORM)

  • Vincent Loreau

Centre National de la Recherche Scientifique

  • Nuno Miguel Luis

Centre National de la Recherche Scientifique

  • Christophe Pitaval

Max-Planck-Gesellschaft

  • Dirk Görlich

Aix-Marseille Université

  • Pierre Mangeol

European Research Council (ERC-2019-SyG 856118)

  • Dirk Görlich

European Research Council (ERC-2019-SyG 856118)

  • Frank Schnorrer

Aix-Marseille Université (A*MIDEX)

  • Frank Schnorrer

Agence Nationale de la Recherche (ANR-11-IDEX-0001-02)

  • Frank Schnorrer

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

Reviewing Editor

  1. Michel Labouesse, UMR7622, Institut de Biologie Paris-Seine, Sorbonne Université, France

Version history

  1. Received: April 7, 2022
  2. Preprint posted: April 15, 2022 (view preprint)
  3. Accepted: December 16, 2022
  4. Accepted Manuscript published: January 16, 2023 (version 1)
  5. Version of Record published: January 30, 2023 (version 2)

Copyright

© 2023, Loreau 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.

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  1. Vincent Loreau
  2. Renate Rees
  3. Eunice HoYee Chan
  4. Waltraud Taxer
  5. Kathrin Gregor
  6. Bianka Mußil
  7. Christophe Pitaval
  8. Nuno Miguel Luis
  9. Pierre Mangeol
  10. Frank Schnorrer
  11. Dirk Görlich
(2023)
A nanobody toolbox to investigate localisation and dynamics of Drosophila titins and other key sarcomeric proteins
eLife 12:e79343.
https://doi.org/10.7554/eLife.79343

Share this article

https://doi.org/10.7554/eLife.79343

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