Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies

Abstract

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15 aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

Article and author information

Author details

  1. Jun Xu

    Department of Genetics, Harvard Medical School, Boston, United States
    For correspondence
    Jun_Xu@hms.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Ah-Ram Kim

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9597-6759
  3. Ross W Cheloha

    Boston Children's Hospital, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Fabian A Fischer

    Boston Children's Hospital, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Joshua Shing Shun Li

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Yuan Feng

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Emily Stoneburner

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Richard Binari

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Stephanie E Mohr

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9639-7708
  10. Jonathan Zirin

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Hidde L Ploegh

    Boston Children's Hospital, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Norbert Perrimon

    Department of Genetics, Harvard Medical School, Boston, United States
    For correspondence
    perrimon@genetics.med.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7542-472X

Funding

National Institute of General Medical Sciences (GM132087)

  • Norbert Perrimon

National Research Foundation of Korea (2021R1A6A3A14039622)

  • Ah-Ram Kim

Croucher Foundation

  • Joshua Shing Shun Li

Howard Hughes Medical Institute

  • Norbert Perrimon

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

Reviewing Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Version history

  1. Preprint posted: April 17, 2021 (view preprint)
  2. Received: September 29, 2021
  3. Accepted: January 24, 2022
  4. Accepted Manuscript published: January 25, 2022 (version 1)
  5. Version of Record published: February 17, 2022 (version 2)

Copyright

© 2022, Xu 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

  • 7,237
    views
  • 1,118
    downloads
  • 21
    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. Jun Xu
  2. Ah-Ram Kim
  3. Ross W Cheloha
  4. Fabian A Fischer
  5. Joshua Shing Shun Li
  6. Yuan Feng
  7. Emily Stoneburner
  8. Richard Binari
  9. Stephanie E Mohr
  10. Jonathan Zirin
  11. Hidde L Ploegh
  12. Norbert Perrimon
(2022)
Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies
eLife 11:e74326.
https://doi.org/10.7554/eLife.74326

Share this article

https://doi.org/10.7554/eLife.74326

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Marian Brenner, Christoph Zink ... Antje Gohla
    Research Article

    Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

    1. Biochemistry and Chemical Biology
    2. Stem Cells and Regenerative Medicine
    Parthasarathy Sampathkumar, Heekyung Jung ... Yang Li
    Research Article

    Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody–RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.