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

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 ProteinsEstablishment of Spatial and Temporal Patterns > GradientsInvertebrate Organogenesis > Flies

Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc

“…Among protein binders against commonly used tags, nanobodies against GFP are used most intensively in the field 49 . However, since tagging with a large tag such as GFP could affect protein functions, as is the case for Dpp, protein binders against small tags have recently been generated to manipulate intracellular proteins [50][51][52][53] .…”

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

“…Combinatorial tagging of POIs would expand the repertoire of protein manipulation. A possibility would be, for example, to use one tag for visualization and the other tag for specific manipulation, as we demonstrated with TkvHAeGFP in vivo (see Aguilar et al, 2019b for a discussion on the use of different tags in the same gene). As pointed out in a recent review, expression levels of the protein binder for visualization of a POI must be carefully controlled for a correct interpretation of the results ( Aguilar et al, 2019b ).…”

“…TkvHAeGFP in vivo (see (Aguilar et al, 2019b) for a discussion on the use of different tags in the same gene). As pointed out in a recent review, expression levels of the protein binder for visualization of a POI must be carefully controlled for a correct interpretation of the results (Aguilar et al, 2019b).…”

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

“…A possibility would be, for example, to use one tag and its specific binder for visualization (with a chromobody) and the other tag for specific manipulation (see next paragraph). As pointed out in a recent review (Aguilar et al, 2019b), expression levels of the protein binder for visualization of a POI must be carefully controlled for a correct interpretation of the results. Strategies such as inducibility (Panza et al, 2015), self-transcriptional autoregulating domain fusion (Son et al, 2016) or intrinsic self stability (Tang et al, 2016), which were developed for nanobodies (Panza et al, 2015; Tang et al, 2016) and fibronectin-derived intrabodies (Son et al, 2016), could be applied to all the small tag binders described here.…”

“…Inserting several tags into an endogenously expressed protein will also allow for efficient recognition of the protein by an antibody against one of the tags, and manipulation of the POI via a functionalized binder recognizing the second tag inserted. The effects of the manipulation can then be followed with antibody staining (see (Aguilar et al, 2019b) for a discussion on the use of several different tags in the same gene).…”