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
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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