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Unintended perturbation of protein function using GFP nanobodies in human cells
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Küey, Cansu, Larocque, Gabrielle, Clarke, Nicholas I. and Royle, Stephen J. (2019) Unintended perturbation of protein function using GFP nanobodies in human cells. Journal of Cell Science, 132 (21). jcs234955. doi:10.1242/jcs.234955
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Official URL: http://dx.doi.org/10.1242/jcs.234955
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.
| Item Type: | Journal Article | |||||||||||||||
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| Subjects: | Q Science > QH Natural history Q Science > QP Physiology |
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| Divisions: | Faculty of Medicine > Warwick Medical School > Biomedical Sciences > Cell & Developmental Biology Faculty of Medicine > Warwick Medical School > Biomedical Sciences Faculty of Medicine > Warwick Medical School |
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| SWORD Depositor: | Library Publications Router | |||||||||||||||
| Library of Congress Subject Headings (LCSH): | Green fluorescent protein, Cell membranes , Endocytosis, Cytology | |||||||||||||||
| Journal or Publication Title: | Journal of Cell Science | |||||||||||||||
| Publisher: | The Company of Biologists | |||||||||||||||
| ISSN: | 1477-9137 | |||||||||||||||
| Official Date: | 10 October 2019 | |||||||||||||||
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| Volume: | 132 | |||||||||||||||
| Number: | 21 | |||||||||||||||
| Article Number: | jcs234955 | |||||||||||||||
| DOI: | 10.1242/jcs.234955 | |||||||||||||||
| Status: | Peer Reviewed | |||||||||||||||
| Publication Status: | Published | |||||||||||||||
| Access rights to Published version: | Open Access | |||||||||||||||
| RIOXX Funder/Project Grant: |
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