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-Tumor Protein D54 (TPD54/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.
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01 January 2019
Unintended perturbation of protein function using GFP nanobodies in human cells
Cansu Küey,
Cansu Küey
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
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Gabrielle Larocque
,
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
,
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
Stephen J. Royle
*
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
*Author for correspondence: s.j.royle@warwick.ac.uk
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Cansu Küey
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
Gabrielle Larocque
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
Nicholas I. Clarke
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
Stephen J. Royle
*
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
*Author for correspondence: s.j.royle@warwick.ac.uk
Received:
31 May 2019
Accepted:
03 Oct 2019
Online ISSN: 1477-9137
Print ISSN: 0021-9533
Medical Research Council
(G070149)
Engineering and Physical Sciences Research Council
(EP/S004459/1)
© 2019. Published by The Company of Biologists Ltd
2019
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
J Cell Sci jcs.234955.
Article history
Received:
31 May 2019
Accepted:
03 Oct 2019
Currently Viewing Accepted Manuscript - Newer Version Available
06 Nov 2019
Citation
Cansu Küey, Gabrielle Larocque, Nicholas I. Clarke, Stephen J. Royle; Unintended perturbation of protein function using GFP nanobodies in human cells. J Cell Sci 2019; jcs.234955. doi: https://doi.org/10.1242/jcs.234955
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