Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would profit from more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we selected a number of short tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POIs binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be utilized for POI manipulation in vivo.

Cellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years.Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time-and spacecontrolled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

Retrograde bone morphogenetic protein (BMP) signaling at the Drosophila neuromuscular junction (NMJ) has served as a paradigm to study TGF-β-dependent synaptic function and maturation. Yet, how retrograde BMP signaling transcriptionally regulates these functions remains unresolved. Here, we uncover a gene network, enriched for neurotransmission-related genes, that is controlled by retrograde BMP signaling in motor neurons through two Smad-binding cis-regulatory motifs, the BMP-activating (BMP-AE) and silencer (BMP-SE) elements. Unpredictably, both motifs mediate direct gene activation, with no involvement of the BMP derepression pathway regulators Schnurri and Brinker. Genome editing of candidate BMP-SE and BMP-AE within the locus of the active zone gene bruchpilot, and a novel Ly6 gene witty, demonstrated the role of these motifs in upregulating genes required for the maturation of pre- and post-synaptic NMJ compartments. Our findings uncover how Smad-dependent transcriptional mechanisms specific to motor neurons directly orchestrate a gene network required for synaptic maturation by retrograde BMP signaling.