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WIREs Dev Biol
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Reflections on the use of protein binders to study protein function in developmental biology

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Abstract Studies in the field of developmental biology aim to unravel how a fertilized egg develops into an adult organism and how proteins and other macromolecules work together during this process. With regard to protein function, most of the developmental studies have used genetic and RNA interference approaches, combined with biochemical analyses, to reach this goal. However, there always remains much room for interpretation on how a given protein functions, because proteins work together with many other molecules in complex regulatory networks and it is not easy to reveal the function of one given protein without affecting the networks. Likewise, it has remained difficult to experimentally challenge and/or validate the proposed concepts derived from mutant analyses without tools that directly manipulate protein function in a predictable manner. Recently, synthetic tools based on protein binders such as scFvs, nanobodies, DARPins, and others have been applied in developmental biology to directly manipulate target proteins in a predicted manner. Although such tools would have a great impact in filling the gap of knowledge between mutant phenotypes and protein functions, careful investigations are required when applying functionalized protein binders to fundamental questions in developmental biology. In this review, we first summarize how protein binders have been used in the field, and then reflect on possible guidelines for applying such tools to study protein functions in developmental biology. This article is categorized under: Technologies > Analysis of Proteins Establishment of Spatial and Temporal Patterns > Gradients Invertebrate Organogenesis > Flies
Schematic representation of the different protein binder‐based methods used in developmental biology. (a) The chromobody approach. Upon binding, protein binder‐FP fusions reveal localization of the POI. (b) LlamaTags. Upon binding to a FP, the POI fused to a protein binder recognizing a FP can be visualized via FP recruitment. (c) deGradFP. Upon binding, the GFP‐nanobody fused to an F‐box domain targets the GFP‐tagged POI for proteasomal degradation. (d) TRIM‐away. An IgG against the POI is introduced into a cell expressing TRIM21. TRIM21 sends the antibody–antigen complex to proteasomal degradation via IgG ubiquitination. (e) JabbaTrap. A GFP nanobody fused to a lipid droplet‐specific scaffold relocalizes GFP‐tagged nuclear proteins to lipid droplets to prevent their normal function. (f) GrabFP. GFP nanobodies fused to different plasma membrane scaffolds (basolateral, apically enriched or homogenously distributed) allow the relocalization of intracellular proteins in different membrane compartment. (g) Morphotrap. A GFP nanobody fused to the transmembrane domain of CD8 allows trapping of secreted molecules on the cell surface and restricting their dispersal. (h) Extracellular GrabFP. A GFP nanobody fused to different plasma membrane scaffolds permits trapping subpopulations of the secreted POI. (i) Nanobody‐kinase. A GFP nanobody fused to a kinase minimal domain induces GFP‐tagged POI phosphorylation
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Recommended experiments to test source‐trapping of secreted ligands. (a) Trapping the POI in the source should eliminate the POI in the target tissue. If the response in the target tissue (signaling, target gene expression, etc.) is also dependent on dispersal, the response should be also eliminated. (b) POI trapped in the receiving cells should be eliminated by efficient trapping the POI in the source. (c) The source trapping phenotype in hemizygotes should not be stronger than that in homozygotes. (d) Phenotypes caused by trapping the POI should resemble those observed by the membrane‐tethered version of POI
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Invertebrate Organogenesis > Flies
Establishment of Spatial and Temporal Patterns > Gradients
Technologies > Analysis of Proteins