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Fluorescent fusion protein knockout mediated by anti-GFP nanobody

Nature Structural & Molecular Biology volume 19, pages 117–121 (2012)Cite this article

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Abstract

The use of genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Recent advances in reverse genetics such as RNA interference and morpholinos are widely used to further apply this paradigm. Nevertheless, such systems act upstream of the proteic level, and protein depletion depends on the turnover rate of the existing target proteins. Here we present deGradFP, a genetically encoded method for direct and fast depletion of target green fluorescent protein (GFP) fusions in any eukaryotic genetic system. This method is universal because it relies on an evolutionarily highly conserved eukaryotic function, the ubiquitin pathway. It is traceable, because the GFP tag can be used to monitor the protein knockout. In many cases, it is a ready-to-use solution, as GFP protein-trap stock collections are being generated in Drosophila melanogaster and in Danio rerio.

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Figure 1: Schematic illustration of deGradFP.
Figure 2: NSlmb-vhhGFP4 mediates degradation of H2B-GFP in mammalian cells and His2Av::EYFP in Drosophila.
Figure 3: Kinetics of target protein degradation by deGradFP in Drosophila.
Figure 4: deGradFP phenocopies loss-of-function mutations.
Figure 5: deGradFP is able to target the intracytoplasmic, but not the extracytoplasmic, part of transmembrane proteins.

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Acknowledgements

We thank A. Santamaria, L. Fava and M. Müller for technical assistance; A. Olichon (Paul Sabatier University) for providing pAOD2-vhhGFP4; Y. Blum (University of Basel, UB) for providing pcDNA3_EGFP-3xNLS; E. Nigg (UB) for providing the HeLa S3 cell line expressing H2B-GFP; B. Bello (UB), W. Gehring (UB), C. Gonzalez (Institute for Research in Biomedicine), R. Karess (Institut Jacques Monod), T. Kornberg (University of California, San Francisco), Y. Hong (University of Pittsburgh), H. Oda (Biohistory Research Hall), R. Schuh (Max Planck Institute for Biophysical Chemistry) and the Bloomington Drosophila stock center (Indiana University), for providing fly stocks. E.C. was supported by an EMBO long-term postdoctoral fellowship (ALTF 737-2005). Work in our laboratory is supported by grants from the Kantons Basel-Stadt and Basel-Land, the Swiss National Science Foundation and the SystemsX.ch initiative within the framework of the WingX project.

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Authors and Affiliations

  1. Biozentrum, University of Basel, Basel, Switzerland

    Emmanuel Caussinus, Oguz Kanca & Markus Affolter

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  1. Emmanuel Caussinus
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Contributions

E.C. created the basic concept of deGradFP, designed all experiments and conducted all experiments except the ones depicted in Figure 4a and Supplementary Figure 3b,c. O.K. generated the ap::GFP flies and conducted the experiments depicted in Figure 4a and Supplementary Figure 3b,c. E.C., O.K. and M.A. wrote the manuscript.

Corresponding authors

Correspondence to Emmanuel Caussinus or Markus Affolter.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Table 1 (PDF 572 kb)

Supplementary Data 1 (RTF 16 kb)

Supplementary Movie 1

deGradFP mediates degradation of His2Av::EYFP in Drosophila. Confocal life imaging of an embryo having the following genotype, ubiHis2Av::EYFP; His2Av::RFP1/enGal4; UAS_NSlmb-vhhGFP4/+. Two channels, corresponding to His2Av::EYFP (green) and His2Av::RFP1 (red), were acquired simultaneously for better time resolution. Lateral views (z–stack maximum projections) were taken at 10 min intervals from stage 6 to 16. Anterior is to the left. Scale bar, 100 μm. (AVI 2570 kb)

Supplementary Movie 2

Wild type dorsal closure.Confocal life imaging of an embryo having the following genotype, sqhAX3; sqhSqh::GFP/+. Sqh::GFP appears in shades of gray. Dorsal views (z–stack maximum projections) were taken at 10 min intervals from stage 12 to 16. Anterior is to the left. Scale bar, 100 μm. (AVI 1851 kb)

Supplementary Movie 3

Sqh::GFP targeting by deGradFP in the amnioserosa cells causes a dorsal open phenotype.Confocal life imaging of an embryo having the following genotype, sqhAX3; sqhSqh::GFP/Gal4332.3; UAS_NSlmb–vhhGFP4/+. Sqh::GFP appears in shades of gray. Dorsolateral views (z–stack maximum projections) were taken at 10 min intervals from stage 12 to 16. Anterior is to the left. Scale bar, 100 μm. (AVI 2008 kb)

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Caussinus, E., Kanca, O. & Affolter, M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat Struct Mol Biol 19, 117–121 (2012). https://doi.org/10.1038/nsmb.2180

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