Abstract
Synthetic protein-binding tools based on anti-green fluorescent protein (GFP) nanobodies have recently emerged as useful resources to study developmental biology. By fusing GFP-targeting nanobodies to well-characterized protein domains residing in discrete sub-cellular locations, it is possible to directly and acutely manipulate the localization of GFP-tagged proteins-of-interest in a predictable manner. Here, we describe a detailed protocol for the application of nanobody-based GFP-binding tools, namely Morphotrap and GrabFP, to study the localization and function of extracellular and intracellular proteins in the Drosophila wing imaginal disc. Given the generality of these methods, they are easily applicable for use in other tissues and model organisms.
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References
Helma J, Cardoso MC, Muyldermans S et al (2015) Nanobodies and recombinant binders in cell biology. J Cell Biol 209:633–644
Harmansa S, Hamaratoglu F, Affolter M et al (2015) Dpp spreading is required for medial but not for lateral wing disc growth. Nature 527:317–322
Bieli D, Alborelli I, Harmansa S et al (2016) Development and application of functionalized protein binders in multicellular organisms. Int Rev Cell Mol Biol 325:181–213
Kaiser PD, Maier J, Traenkle B et al (2014) Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells. Biochim Biophys Acta 1844:1933–1942
Plückthun A (2015) Designed ankyrin repeat proteins (DARPins): Binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol 55:489–511
Aguilar G, Matsuda S, Vigano MA et al (2019) Using nanobodies to study protein function in developing organisms. Antibodies (Basel) 8:16
Aguilar G, Vigano MA, Affolter M et al (2019) Reflections on the use of protein binders to study protein function in developmental biology. Wiley Interdiscip Rev Dev Biol 8:e356
Saerens D, Pellis M, Loris R et al (2005) Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol 352:597–607
Rothbauer U, Zolghadr K, Muyldermans S et al (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7:282–289
Matsuda S, Harmansa S, Affolter M (2016) BMP morphogen gradients in flies. Cytokine Growth Factor Rev 27:119–127
Matsuda S, Affolter M (2017) Dpp from the anterior stripe of cells is crucial for the growth of the Drosophila wing disc. eLife 6:e22319
Harmansa S, Alborelli I, Bieli D et al (2017) A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila. eLife 6:e22549
Greenspan RJ (2004) Fly pushing: the theory and practice of Drosophila genetics, 2nd edn. CSHL Press, Cold Spring Harbor
Morin X, Daneman R, Zavortink M et al (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci U S A 98:15,050–15,055
Kelso RJ, Buszczak M, Quiñones AT et al (2004) Flytrap, a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster. Nucleic Acids Res 32:D418–D420
Buszczak M, Paterno S, Lighthouse D et al (2007) The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175:1505–1531
Asakawa K, Kawakami K (2009) The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish. Methods 49:275–281
Kawakami K, Abe G, Asada T et al (2010) zTrap: Zebrafish gene trap and enhancer trap database. BMC Dev Biol 10:105
Sarov M, Barz C, Jambor H et al (2016) A genome-wide resource for the analysis of protein localisation in Drosophila. eLife 5:e12068
Ringrose L (2009) Transgenesis in Drosophila melanogaster. Methods Mol Biol 21:3–19
Venken KJT, Bellen HJ (2007) Transgenesis upgrades for Drosophila melanogaster. Development 134:3571–3584
Spratford CM, Kumar JP (2014) Dissection and immunostaining of imaginal discs from Drosophila melanogaster. J Vis Exp 91:51792
Blair SS (2007) Dissection of imaginal discs in Drosophila. Cold Spring Harb Protoc 2007:pdb.prot4794
Matsuda, S., Schaefer, J.V., Mii, Y. et al. Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc. Nat Commun 12, 6435 (2021).
Vigano MA, Ell C-M, Kustermann MMM et al (2021) Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster. Development 148:dev191700
Yagi R, Mayer F, Basler K (2010) Refined LexA transactivators and their use in combination with the Drosophila Gal4 system. Proc Natl Acad Sci U S A 107:16,166–16,171
Kanca O, Caussinus E, Denes AS et al (2014) Raeppli: a whole-tissue labeling tool for live imaging of Drosophila development. Development 141:472–480
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Matsuda, S., Aguilar, G., Vigano, M.A., Affolter, M. (2022). Nanobody-Based GFP Traps to Study Protein Localization and Function in Developmental Biology. In: Hussack, G., Henry, K.A. (eds) Single-Domain Antibodies. Methods in Molecular Biology, vol 2446. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2075-5_30
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DOI: https://doi.org/10.1007/978-1-0716-2075-5_30
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