Cpx nitrosylation and block of farnesylation leads to redistribution of GFP-CAAX and enhances Dmcpx localization at AZs.
(A) GFP-CAAX expression in HEK cells showing membrane fluorescence signals in Ctrls. Pharmacological inhibition of farnesylation by GGTI-298 + L-744,832 (“farnesyl inh,” p < 0.0001 versus Ctrl, ANOVA) and NO incubation (p < 0.0001 versus Ctrl, ANOVA) result in a redistribution of GFP fluorescence into the cytosol. Fluorescence signals were analyzed by line scan and plotted as intensities (a.u.) over distance across the cell somata, n = 80–104 cells, scale bar: 20 μm. (B) Mouse cpx-3 is S-nitrosylated in response to NO donor application. Immunoblot intensities increased 2.4 ± 0.5-fold following NO application. (C) Representative recordings of a 50-Hz train and spontaneous activity of a larva expressing Dmcpx 7AC140W with mean eEJC amplitudes, QC, vesicle pool size, and mEJC frequency shown in (D). (E) Top, maximal projection confocal images of NMJs expressing a WT cpx or Dmcpx 7AC140W showing the PLA signal in red; bottom, STED images showing cpx and Brp staining in WT and Dmcpx 7AC140W mutants. (F) Analysis of PLA data. (G) Co-localization data with Pearson’s coefficient for interactions of cpx with Brp. The raw data can be found in S8 Data. Data denote mean ± SEM, Student t test (D, F), *p < 0.05, ***p < 0.001, ****p < 0.0001. a.u., arbitrary unit; AZ, active zone; Brp, Bruchpilot; cpx, complexin; Ctrl, control; eEJC, evoked EJC; EJC, excitatory junction current; GFP, green fluorescent protein; HEK, human embryonic kidney; HRP, horseradish peroxidase; mEJC, miniature EJC; NMJ, neuromuscular junction; NO, nitric oxide; PLA, proximity ligation assay; QC, quantal content; STED, stimulated emission depletion; WT, wild-type.