zGrad is a nanobody-based degron system that inactivates proteins in zebrafish

  1. Naoya Yamaguchi
  2. Tugba Colak-Champollion
  3. Holger Knaut  Is a corresponding author
  1. New York University School of Medicine, United States

Abstract

The analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane, cytosolic and nuclear proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Two main tables are provided: Table 1 as a detail numerical data set for supporting Cdh1 transgene is fully functional; Table 2 as detail numerical data set for protein degradation kinetics exported from fitting against the one-exponential decay model. Source code files 1-3 describe the custom Image J macros written to analyze data. The pCS2-zGrad plasmid is available from Addgene (https://www.addgene.org/119716/). Transgenic fish lines are available from our lab upon request to the corresponding author.

Article and author information

Author details

  1. Naoya Yamaguchi

    Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Tugba Colak-Champollion

    Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Holger Knaut

    Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, United States
    For correspondence
    Holger.Knaut@med.nyu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8399-8720

Funding

Eunice Kennedy Shriver National Institute of Child Health and Human Development (R21HD088779)

  • Holger Knaut

National Institute of Neurological Disorders and Stroke (R01NS102322)

  • Holger Knaut

NYSTEM (C322560GG)

  • Naoya Yamaguchi

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

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (IA16-00788_AMEND3) of the NYU School of Medicine.

Reviewing Editor

  1. Markus Affolter, Biozentrum der Universität Basel, Switzerland

Version history

  1. Received: October 25, 2018
  2. Accepted: February 7, 2019
  3. Accepted Manuscript published: February 8, 2019 (version 1)
  4. Version of Record published: February 21, 2019 (version 2)

Copyright

© 2019, Yamaguchi 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. Naoya Yamaguchi
  2. Tugba Colak-Champollion
  3. Holger Knaut
(2019)
zGrad is a nanobody-based degron system that inactivates proteins in zebrafish
eLife 8:e43125.
https://doi.org/10.7554/eLife.43125

Share this article

https://doi.org/10.7554/eLife.43125

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