Figure 6.

DNRX interacts with DPix through Scribble and regulates the activity of Rac1, and maintains the terminal SV aggregation and release. A, Western blots of anti-GFP immunoprecipitates using protein lysates of adult Drosophila heads showing immunoprecipitated proteins for DNRX and DPix in vivo. Input is 1%. B, Western blots of anti-GFP immunoprecipitates using protein lysate of adult Drosophila heads showing loss of Scribble, DNRX could not immunoprecipitate with DPix in vivo. Input is 1%. C, schematic representation of DNRX interacts with DPix through Scribble and activates Rac1. D, Western blots showing the level of Rac-GTP and total Rac1 in adult heads of wild-type, dnrx mutant. E, summary graph showing that the activated Rac1 was decreased after knocking out DNRX. F, Western blots showing the level of Rac-GTP and total Rac1 in adult heads of elav dcr/+, dnrx, and scribble knocking down flies. G, summary graph showing that the activated Rac1 was decreased after knocking down the expression of DNRX and Scribble using the RNAi method. H, summary graph showing that the mean fluorescence intensity of DPix-GFP was decreased both in NMJ and VNC in dnrx mutant when compared with wild-type controls. I and J″, representative images of larvae VNC of the indicated genotypes were stained with DNRX (red) and GFP (green), showing DNRX regulates the amount of DPix. K and L‴, representative images of larvae NMJ type Ib boutons of the indicated genotypes were stained with HRP (blue), DLG (red), and GFP (green), showing DNRX regulates the amount of DPix. M–O″, confocal images of third instar larvae NMJ type Ib boutons of the indicated genotypes double labeled with anti-SYT (red) and anti-HRP (green), which labels the pre-synaptic SVs and neuronal membrane, respectively. P, quantification of the ratio of the SYT-diffused boutons in muscle 4 shows that in dnrx mutants the distribution of SVs has been disturbed and this phenotype can be rescued by pre-synaptic DPix. Q, representative traces of spontaneous responses of the indicated genotypes. R, quantification of mEJP frequency of indicated genotypes, showing that mEJP frequency was increased in the dnrx mutant and the Scribble and DPix could rescue the defects at pre-synapse. Data are mean ± S.E., ***, p < 0.001; **, p < 0.01; and *, p < 0.05. ns, not significant. Two-tailed Student's t tests were used to compare genotypes. Scale bars, 50 (I–J″), 5 (K-L‴), and 5 μm (M–O″).