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TOOLS AND RESOURCES
Unintended perturbation of protein function using GFP nanobodies in human cells
Cansu Küey, Gabrielle Larocque, Nicholas I. Clarke, Stephen J. Royle
Journal of Cell Science 2019 132: jcs234955 doi: 10.1242/jcs.234955 Published 6 November 2019
Cansu Küey
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
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  • ORCID record for Cansu Küey
Gabrielle Larocque
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
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Nicholas I. Clarke
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
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Stephen J. Royle
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
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ABSTRACT

Tagging a protein of interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed ‘dongles’, that could add, for example, an FKBP tag to a GFP-tagged protein of interest, enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2–GFP caused inhibition of dynamin function prior to knocksideways. The function of GFP-tagged tumor protein D54 (TPD54, also known as TPD52L2) in anterograde traffic was also perturbed by dongles. While these issues potentially limit the application of dongles, we discuss strategies for their deployment as cell biological tools.

This article has an associated First Person interview with the first author of the paper.

Footnotes

  • Competing interests

    The authors declare no competing or financial interests.

  • Author contributions

    Conceptualization: N.I.C., S.J.R.; Methodology: C.K., N.I.C.; Validation: C.K.; Formal analysis: C.K., G.L., S.J.R.; Investigation: C.K., G.L.; Resources: N.I.C.; Writing - original draft: S.J.R.; Writing - review & editing: C.K., G.L., S.J.R.; Visualization: C.K.; Supervision: G.L., S.J.R.

  • Funding

    The work was supported by the UK Medical Research Council (MR/P018947/1). C.K. was supported by University of Warwick, and the Engineering and Physical Sciences Research Council and Biotechnology and Biological Sciences Research Council Centre for Doctoral Training in Synthetic Biology (grant EP/L016494/1). Deposited in PMC for immediate release.

  • Supplementary information

    Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.234955.supplemental

  • Received May 31, 2019.
  • Accepted October 3, 2019.
  • © 2019. Published by The Company of Biologists Ltd
http://creativecommons.org/licenses/by/4.0

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Keywords

  • Clathrin-mediated endocytosis
  • Dynamin
  • Nanobody
  • GFP-binding protein
  • Knocksideways

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TOOLS AND RESOURCES
Unintended perturbation of protein function using GFP nanobodies in human cells
Cansu Küey, Gabrielle Larocque, Nicholas I. Clarke, Stephen J. Royle
Journal of Cell Science 2019 132: jcs234955 doi: 10.1242/jcs.234955 Published 6 November 2019
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TOOLS AND RESOURCES
Unintended perturbation of protein function using GFP nanobodies in human cells
Cansu Küey, Gabrielle Larocque, Nicholas I. Clarke, Stephen J. Royle
Journal of Cell Science 2019 132: jcs234955 doi: 10.1242/jcs.234955 Published 6 November 2019

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