Fig. 6: Cytosolic Nb-mScarlet captures Nrx-1-AT co-localization with Rab2 vesicles in the soma and trafficking along the motor neuron axons.
From: Versatile nanobody-based approach to image, track and reconstitute functional Neurexin-1 in vivo
a, b Live imaging of Nrx-1-AT trafficking along the motor neuron axons. Representative snapshots and kymographs from time-lapse confocal imaging of axons co-expressing UAS-Nb-mScarlet with either UAS-Nrx-1-AT (a) or UAS-Nrx-1 (b) under the control of Nrx-1-Gal4. No mScarlet-positive puncta/vesicles are detectable in the absence of the ALFA tag. Vesicles move away (green) or towards the VNC (red) with similar mean velocities (c). N = 18 individual larvae were imaged over three different days; moving vesicles were detected and captured in two or three individual bundles per animal. Data are represented as mean ± SEM (unpaired t test). 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. d–h Representative confocal images of VNC from third instar larvae expressing Nrx-1-AT, Nb-mScarlet, and Rab2CA-YFP under the control of Nrx-1-Gal4. The specimens were fixed and labeled for mScarlet (magenta), YFP (cyan), and HRP (yellow). Maximum projections of the z-planes containing the motor neurons soma are shown and the regions of interest comprising several motor neurons are marked. Vesicles identified by segmentation are shown in the lower panels and in the expanded Supplementary Fig. 17. Overlap of HRP-positive puncta on the combined mScarlet- and Rab2-positive puncta (f”) highlights the composition of HRP-positive puncta. g Scatter plot indicating mScarlet and Rab2 intensity in each HRP puncta enabled the analysis of puncta composition (h) 45% of the HRP puncta (614/1355 total) are positive for both mScarlet and Rab2. Linear regression and non-linear fit tests were used. The null hypothesis was tested using an F-test: F = 53.02 (Df = 1, Dfd = 1353), slope (0.1306 − 0.2269), Y-intercept (0.1507 − 0.1913) 95% confidence, p < 0.0001. i–m Confocal images and puncta analysis in VNCs from third instar larvae expressing untagged Nrx-1, Nb-mScarlet, and Rab2CA-YFP under the control of Nrx-1-Gal4. The analyses followed the workflow described above and revealed only 0.37% overlap among HRP, mScarlet, and Rab2 (5 vesicles /1343 total). F-test: F = 0.501 (Dfn = 1, Dfd = 1341), slope (− 0.006971 − 0.01484), Y-intercept (− 0.006971 − 0.01484) 95% confidence, p = 0.4331. Scale bars: 10 µm. Source data are provided as a Source Data file.
