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.
Highlights
Despite of its importance, the role of protein localization and the effects of forced protein mislocalization have not been studied extensively and remain in many cases not well understood
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
Our results suggest that the functional Dpp morphogen gradient forms in the lateral plane of the wing disc epithelium
Summary
The role of protein localization and the effects of forced protein mislocalization have not been studied extensively and remain in many cases not well understood. While protein function was largely studied by genetic manipulation at the DNA or RNA levels in the past, protein binders allow direct, specific and acute modification and interference of protein function in vivo (Kaiser et al, 2014; Bieli et al, 2016) and might represent valid tools to study protein localization. VhhGFP4 functions in the intracellular environment and can be fused to other proteins without losing its activity and specificity in vivo (Rothbauer et al, 2008). VhhGFP4 has been functionalized by fusing it to different protein domains in order to visualize (Rothbauer et al, 2006), relocalize (Berry et al, 2016) and degrade (Caussinus et al, 2012; Shin et al, 2015) GFP-tagged proteins of interest. GFP nanobodies were used to generate inducible tools that allow controlled transcription (Tang et al, 2013) and enzyme activity (Tang et al, 2015), and to generate synthetic receptors (Harmansa et al, 2015; Morsut et al, 2016), to mention only a few examples
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