Fig. 1: ALFA tag site selection and in vivo detection.

a Domain organization of the Drosophila Nrx-1. The transgenes utilized in this study encode the predominant isoform (Nrx-1-RA) and the corresponding ALFA-tagged and GFP-tagged variants. b Alignment of Nrx-1 intracellular domains from various insect species indicates several blocks of conservation, including the C-terminal PDZ binding motif and the Diptera-specific G-rich expansions. The ALFA-tag insertion site is marked, together with other interaction domains of Drosophila Nrx-1: The Ephrin binding domain (residues 1760–1813) and the NSF binding site (residues 1788–1813). (Dm- Drosophila melanogaster, Md- Musca domestica, Cc- Ceratitis capita, Rc- Rhagoletis cerasi, Tc- Tribolium castaneum, Bm- Bombyx mori, Dp- Danaus Plexippus, Am- Apis mellifera, Hs- Harpegnathos saltator). c–j Representative confocal images of third instar larvae VNC (c, d, e, g and i) and NMJs (f, h, and j) of the indicated genotypes labeled for Nrx-1 (magenta), GFP (green), and ALFA tag (cyan) showing the distribution of endogenous Nrx-1 and neuronally expressed Nrx-1 variants. These experiments were repeated three times with similar results. When overexpressed in the motor neurons, Nrx-1 decorates the presynaptic membrane and accumulates in distinct puncta at synaptic terminals. These puncta coincide with the ALFA-positive signals. In contrast, the GFP-positive signals are more diffuse and separated from the Nrx-1-positive puncta. The GFP and ALFA tag signals/channels are shown for all genotypes to illustrate the background signal. Scale bars: 10 µm. Genotypes: control (w¹¹¹⁸), MN > UAS-Nrx-1 ^{tag/no tag} (BG380-Gal4/+; UAS-Nrx-1 ^{tag/no tag}/+), Nrx-1^null (Nrx²⁷³/Df).