A nanobody toolbox to investigate localisation and dynamics of Drosophila titins and other key sarcomeric proteins
- Aix Marseille University, CNRS, IDBM, France
- 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
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.
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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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
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