A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

Abstract

The role of protein localization along the apical-basal axis of polarized cells is difficult to investigate in vivo, partially due to lack of suitable tools. Here, we present the GrabFP system, a collection of four nanobody-based GFP-traps that localize to defined positions along the apical-basal axis. We show that the localization preference of the GrabFP traps can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization. These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization. Furthermore, we used the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.DOI: http://dx.doi.org/10.7554/eLife.22549.001

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

“…Targeting proteins to a specific location using nanobodies has been applied to study proteinprotein interaction in cells (Herce, Deng et al, 2013) and Drosophila (Harmansa, Alborelli et al, 2017). Herce et al fused an anti-GFP nanobody to the lac repressor to localize the nanobody and its GFP-fused interaction partner to the nucleus.…”

“…Using this approach, the authors have studied binding and disruption of p53 and HDM2 (human double minute 2), one of the most important protein interactions in cancer research (Herce et al, 2013). Harmansa et al have mislocalized transmembrane proteins, cytosolic proteins, and morphogens in Drosophila to study the role of correct protein localization for development in vivo (Harmansa et al, 2017). Targeting to organelles using nanobodies has also been achieved for two specific proteins, p53 and survivin (Beghein, Van Audenhove et al, 2016, Steels, Verhelle et al, 2018.…”

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

“…To trap GFP-tagged secreted POIs on the cell surface, a GFP nanobody has been fused to the transmembrane protein CD8 ("morphotrap", Harmansa, Hamaratoglu, Affolter, & Caussinus, 2015). The GFP nanobody has also been fused to basolaterally localized or apically enriched scaffolds to trap subpopulations of GFP-tagged POI extracellularly, thereby interfering with different pools of GFP-tagged secreted POI (Harmansa et al, 2017), (Figure 1e).…”

“…In addition, and since POIs may also have strong regulation of localization, the GrabFPs can be relocalized by the POI (instead of the other way around). In this case, relocalization of the POI to the expected destination may not be possible by these methods (Harmansa et al, 2017). So far, almost all the relocalizing tools have been generated with the VHH4 anti GFP nanobody (See Table 1).…”

Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos

“…GBPs fused with subcellular localization signals can be used to identify protein-protein interactions with tagged bait, or to mislocalize target GFP-tagged proteins (reviewed in Harmansa & Affolter, 2018). The GrabFP system (Harmansa et al, 2017) redirects target proteins to set positions within the apical-basal axis of epithelial cells, for example. A similar “morphotrap” method couples GBP nanobodies to the extracellular surface of the cell, where they act to immobilize secreted GFP-tagged bait (Harmansa et al, 2015).…”