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.
Reviewing Editor
- Michel Labouesse, UMR7622, Institut de Biologie Paris-Seine, Sorbonne Université, France
Version history
- Received: April 7, 2022
- Preprint posted: April 15, 2022 (view preprint)
- Accepted: December 16, 2022
- Accepted Manuscript published: January 16, 2023 (version 1)
- 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.
Metrics
-
- 1,405
- Page views
-
- 238
- Downloads
-
- 3
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
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)
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
Further reading
-
- Developmental Biology
- Stem Cells and Regenerative Medicine
The attachment site of the rotator cuff (RC) is a classic fibrocartilaginous enthesis, which is the junction between bone and tendon with typical characteristics of a fibrocartilage transition zone. Enthesis development has historically been studied with lineage tracing of individual genes selected a priori, which does not allow for the determination of single-cell landscapes yielding mature cell types and tissues. Here, in together with open-source GSE182997 datasets (three samples) provided by Fang et al., we applied Single-cell RNA sequencing (scRNA-seq) to delineate the comprehensive postnatal RC enthesis growth and the temporal atlas from as early as postnatal day 1 up to postnatal week 8. And, we furtherly performed single-cell spatial transcriptomic sequencing on postnatal day 1 mouse enthesis, in order to deconvolute bone-tendon junction (BTJ) chondrocytes onto spatial spots. In summary, we deciphered the cellular heterogeneity and the molecular dynamics during fibrocartilage differentiation. Combined with current spatial transcriptomic data, our results provide a transcriptional resource that will support future investigations of enthesis development at the mechanistic level and may shed light on the strategies for enhanced RC healing outcomes.
-
- Developmental Biology
- Neuroscience
Development of the nervous system depends on signaling centers – specialized cellular populations that produce secreted molecules to regulate neurogenesis in the neighboring neuroepithelium. In some cases, signaling center cells also differentiate to produce key types of neurons. The formation of a signaling center involves its induction, the maintenance of expression of its secreted molecules, and cell differentiation and migration events. How these distinct processes are coordinated during signaling center development remains unknown. By performing studies in mice, we show that Lmx1a acts as a master regulator to orchestrate the formation and function of the cortical hem (CH), a critical signaling center that controls hippocampus development. Lmx1a co-regulates CH induction, its Wnt signaling, and the differentiation and migration of CH-derived Cajal–Retzius neurons. Combining RNAseq, genetic, and rescue experiments, we identified major downstream genes that mediate distinct Lmx1a-dependent processes. Our work revealed that signaling centers in the mammalian brain employ master regulatory genes and established a framework for analyzing signaling center development.
