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. Author manuscript; available in PMC: 2022 Dec 6.
Published in final edited form as: Dev Cell. 2021 Nov 5;56(23):3235–3249.e4. doi: 10.1016/j.devcel.2021.10.011

Figure 7: UNC-4 promotes gap junction assembly in VA axon during larval development.

Figure 7:

(A) AVA::UNC-7::tagRFP (magenta) and VA::GFP::UNC-9 (green) and co-localized UNC-7 and UNC-9 puncta (white) in VA axons of wild-type and unc-4 mutant L2 larvae. Scale bar = 2.5 μm.(B) Violin plots for density of co-localized UNC-7::tagRFP and GFP::UNC-9 puncta in wild-type and unc-4 VAs in L2 and L4 larvae. L4 data for wild type and unc-4 from Figure 4C. Dashed line represents median, 2-way ANOVA, **** = p < 0.0001. NS = Not Significant. (C) Schematic of FRAP strategy. A single VA axon (VA2, VA3, or VA4) is bleached in individual L3 larvae. Each treated animal is re-imaged 24-hours later (young adult) to record fluorescence intensity from photobleached VA axon. VA::GFP::UNC-9 puncta in (D) wild type and (E) unc-4 in larvae (L3) before photobleaching and at the young adult (YA) stage. Dashed outline denotes VA soma. Scale bars = 2.5 μm. GFP::UNC-9 in soma of unc-4 mutant VA (arrowheads). White line denotes unc-4 axon showing little fluorescence recovery. (F) Relative fluorescence intensity of individual VAs immediately after photobleaching (Bleach) and after 24 hours (Recovery) in wild type and unc-4. Intensity values are normalized to VA axon length and to fluorescence prior to bleaching. 2-way ANOVA, ** p = 0.0023. NS = Not Significant. (G) UNC-4 maintains cAMP to direct trafficking, neuron-specific assembly, subcellular placement and developmental scaling of electrical synapses. (Left) In wild-type VAs, UNC-4 blocks expression of cAMP antagonists, PDE-1 (phosphodiesterase) and FRPR-17(GOA-1-coupled GPCR), to maintain cAMP. cAMP promotes trafficking of UNC-9 in the VA axon for assembly with UNC-7 at heterotypic VA→AVA electrical synapses. Additional UNC-9 puncta accumulate in the wild-type VA axon during larval development. (Right) Ectopic expression of PDE-1 and FRPR-17 in unc-4 mutant VAs reduces cAMP levels and limits UNC-9 trafficking. UNC-9 accumulates in VA soma for assembly with UNC-7 at heterotypic VA→AVB gap junctions.