Background

The range of intercellular dispersion and signaling activity of many morphogenetic signaling proteins depends on 'limited proteolysis' by proprotein convertases (PCs) (LeMosy, 2006; Künnapuu et al., 2009; Wharton and Serpe, 2013; Sohr et al., 2019). PCs are serine endoprotease family enzymes that are known to cleave various growth factors including Epidermal growth factor (EGF), the Transforming Growth Factor beta/Bone Morphogenetic Protein (TGF-β/BMP) family, several Fibroblast Growth Factor (FGF) family proteins, Platelet-Derived Growth Factor (PDGF), and Vascular Endothelial Growth Factor (VEGF), all of which play a critical role in development and diseases (Khatib and Geraldine, 2006; Tulin and Stathopoulos, 2010; Rousselet et al., 2011; Künnapuu et al., 2014; Constam, 2014; Anderson and Wharton, 2017; Sohr et al., 2019). Although biochemical and cell culture assays are sufficient to demonstrate a substrate cleavage by individual PC substrate-specificity, their functional characterization in vivo is largely dependent on classical genetic approaches. However, the presence of multiple PCs in different organisms and their functional redundancy can only provide limited information about an individual PC's function. There are nine PCs in vertebrates and three in Drosophila, all expressed broadly, that share a consensus cleavage site (-Arg-X-Lys/Arg-Arg-) in their substrates. Moreover, a broad range of cellular substrates as well as extracellular matrix proteins that can also influence a signaling or morphogenetic outcome makes the genetic results hard to interpret.

Here we present a protocol that enables direct visualization of Furin-dependent signal cleavage by monitoring the spatiotemporal fates of the cleaved portions of a substrate in developing organs. The strategy and methodology was designed to examine Drosophila Bnl cleavage by Furin. The experiment takes advantage of the 3rd instar larval wing imaginal disc and an associated tracheal branch called the air sac primordium (ASP) (Sato and Kornberg, 2002). Bnl is expressed by a restricted group of wing disc cells and subsequently transported to the ASP to control its morphogenesis (Sato and Kornberg, 2002; Du et al., 2018). Bnl is cleaved by Furin-1 at the 164^th amino acid residue prior to the interorgan transport of only its C-terminal truncated portion that contains the receptor binding site (Sohr et al., 2019). The truncated part of Bnl forms a characteristic long-range gradient in the ASP by binding to its receptor and traveling through actin-based filopodia or cytonemes extended by ASP cells (Du et al., 2018). To visually monitor the fates of the full-length Bnl and its truncated portions, we first generated a transgenic Drosophila line harboring a chimeric Bnl construct with an HA-tag at the 87^th and a GFP tag at 432^nd residue, flanking the cleavage site and conserved FGF domain. The construct was expressed in the wing disc Bnl source. Laval wing discs were dissected and ex vivo-cultured in the absence or presence of Furin protease inhibitors for variable amounts of time followed by α-HA immunostaining and fluorescence microscopic imaging of the discs. While in normal conditions only the C-terminal GFP-tagged fragment of Bnl was transported to the ASP, the addition of Furin inhibitors to the ex vivo culture resulted in uncleaved Bnl with both tags being transported to the recipient ASP cells (Figure 1). Since a broad range of signaling proteins involved in morphogenetic and disease pathways are modulated by Furin, the methodology described here can be a powerful tool to evaluate the role of Furin cleavage in various other signaling pathways. For instance, despite the wide-spread occurrence of signal cleavage modulating paracrine signaling activity, most full-length uncleaved forms of the signals were also found to activate receptors and are shown to be secreted when expressed in cultured cells (Künnapuu et al., 2009; Tulin and Stathopoulos, 2010; Sopory et al., 2010; Tokhunts et al., 2010; Constam, 2014). Therefore, a link between PC-dependent cleavage with the maturation and activity of the proteins remained obscure. The results from this assay together with other experiments showed that uncleaved Bnl is active and can be transported to the recipient cells to induce signaling. However, Bnl cleavage ensures its efficient polarized target-specific dispersion, thereby modulating its range and tissue-specific activity (Sohr et al., 2019). The ex vivo culture method with pharmacological Furin inhibition strategy is not limited to only the Drosophila wing imaginal disc and ASP tissue system. Similar assays can easily be applied to other Drosophila tissues, signaling proteins, or model systems.