Fig. 5: Compartment specific detection using genetically encoded cytosolic Nb-mScarlet.

a–e Representative confocal images of VNCs (a, c) and NMJs (d, f) from third instar larvae expressing cytosolic Nb-mScarlet together with either untagged Nrx-1 or Nrx-1-AT, as indicated. All transgenes were expressed under the control of Nrx-1-Gal4, and the specimens were fixed and labeled for Nrx-1 (green) and mScarlet (magenta). These experiments were repeated three times with similar results; n = 7 (a–c) or 14 (d–f) per genotype. Both Nrx-1 and Nrx-1-AT distribute to synaptic terminals. To quantify the co-localization between Nrx-1 and mScarlet channels, Pearson’s coefficient was calculated within an HRP-selected mask (b, e). In the absence of an ALFA tag, Nb-mScarlet does not co-localize with Nrx-1 and instead localizes to the neuron soma (see also Supplementary Fig. 10) and throughout the synaptic boutons. In larvae expressing Nrx-1-AT, Nb-mScarlet mirrors the Nrx-1 distribution. (g–j) Representative confocal images of larval sensory neurons from control (g) or Nrx-1-AT/+ heterozygous (i) third instar larvae expressing cytosolic Nb-mScarlet and membrane-bound mCD4-GFP under the control of ppk-Gal4. These experiments were repeated three times with similar results; n = 7 for each genotype. The fillets were fixed and labeled for mScarlet (magenta), GFP (green), and HRP (blue). The mScarlet signals (shown in Fire Lut) were further filtered using Gaussian blur and image subtraction (h, j). Multiple mScarlet-positive vesicles, marked by yellow arrows, are detected in larvae with one copy of Nrx-1-AT but not in control. Additional examples are shown in Supplementary Fig. 14. Scale bars: 10 µm. Data are represented as mean ± SEM (unpaired t test); ****p < 0.0001. 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.