A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in brain neurons

  1. Jie-Xian Dong
  2. Yongam Lee
  3. Michael Kirmiz
  4. Stephanie Palacio
  5. Camelia Dumitras
  6. Claudia M Moreno
  7. Richard Sando
  8. L Fernando Santana
  9. Thomas C Südhof
  10. Belvin Gong
  11. Karl D Murray
  12. James S Trimmer  Is a corresponding author
  1. University of California, Davis, United States
  2. University of Washington, United States
  3. Stanford School of Medicine, United States

Abstract

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Jie-Xian Dong

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Yongam Lee

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Michael Kirmiz

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Stephanie Palacio

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Camelia Dumitras

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Claudia M Moreno

    Department of Physiology and Biophysics, University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Richard Sando

    Department of Molecular and Cellular Physiology, Stanford School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. L Fernando Santana

    Department of Physiology and Membrane Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4297-8029
  9. Thomas C Südhof

    Department of Molecular and Cellular Physiology, Stanford School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Belvin Gong

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Karl D Murray

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. James S Trimmer

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    For correspondence
    jtrimmer@ucdavis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6117-3912

Funding

National Institutes of Health (U01NS099714)

  • James S Trimmer

National Institutes of Health (U24NS109113)

  • James S Trimmer

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

Ethics

Animal experimentation: All procedures involving llamas were performed at Triple J Farms of Kent Laboratories (Bellingham, WA) in strict accordance with the Guide for the Care and Use of Laboratory Animals of the NIH. All procedures involving rats were approved by the University of California, Davis, Institutional Animal Care and Use Committee (IACUC) under protocols 20485 and 21265 and were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the NIH. All rats were maintained under standard light-dark cycles and allowed to feed and drink ad libitum. All procedures involving mice were approved by the Stanford University IACUC under protocol 18846 and were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the NIH.

Reviewing Editor

  1. Graeme W Davis, University of California, San Francisco, United States

Publication history

  1. Received: May 24, 2019
  2. Accepted: September 18, 2019
  3. Accepted Manuscript published: September 30, 2019 (version 1)
  4. Version of Record published: October 9, 2019 (version 2)

Copyright

© 2019, Dong 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. Jie-Xian Dong
  2. Yongam Lee
  3. Michael Kirmiz
  4. Stephanie Palacio
  5. Camelia Dumitras
  6. Claudia M Moreno
  7. Richard Sando
  8. L Fernando Santana
  9. Thomas C Südhof
  10. Belvin Gong
  11. Karl D Murray
  12. James S Trimmer
(2019)
A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in brain neurons
eLife 8:e48750.
https://doi.org/10.7554/eLife.48750
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