Fig. 3: Detection and in vivo reconstitution of endogenously edited ALFA-tagged Nrx-1.

a, b Representative traces of mEJPs and EJPs in control and endogenously tagged Nrx-1^AT third instar larvae. c–e Quantification of mEJP amplitude, EJP amplitude, and Quantal Content in the indicated genotypes; n = 9 for the control and 10 for all the other genotypes. f, i Representative confocal images of VNCs or muscle 6/7 NMJs of third instar larvae of the indicated genotypes labeled for ALFA tag (green) and Nrx-1 or HRP (magenta). The images were acquired with the same confocal microscopy settings and scaled equally for direct visual comparison. These experiments were repeated three times with similar results; n = 15 for (f–j) and 5 for (i). The edited Nrx-1^AT shows the expected Nrx-1 accumulation in neurites. j, k Representative traces of mEJPs and EJPs recorded in Nrx^ΔPDZ-AT third instar larvae alone (j) or in the presence of neuronally expressed Nb-PDZ chimera (k). Deletion of the PDZ binding motif of Nrx-1 causes loss-of-function phenotypes that resemble the Nrx-1^null mutant deficits. These phenotypes are completely rescued by neuronal expression of the Nb-PDZ chimera. l–o Representative confocal images of VNCs or muscle 6/7 NMJs of third instar larvae of the indicated genotypes labeled for ALFA tag (green) and Nrx-1 or HRP (magenta). These experiments were repeated three times with similar results; n = 15 for (l–n) and 5 for (o). The absence of a PDZ binding motif shifts the distribution of Nrx^ΔPDZ-AT from the neurites to the motor neuron soma, more specifically in the ER. The addition of the PDZ binding motif in trans restores the cell surface location and normal distribution of reconstituted Nrx-1 (Nrx^ΔPDZ-AT/Nb-PDZ) to the neuropil. Scale bars: 10 µm. Data are represented as mean ± SEM (one-way ANOVA with Tukey’s multiple comparisons); ****p < 0.0001, **p < 0.005, ns, p > 0.05. The boxes expand from the first to the third quartile, and the whiskers from minimum to maximum values; the center lines mark the mean values. Source data are provided as a Source Data file. Genotypes: control (w¹¹¹⁸), Nrx^ΔPDZ-AT; MN > Nb-PDZ (BG380-Gal4/+; UAS-Nb-PDZ/+; Nrx^ΔPDZ-AT).