Figure 4. Synaptojanin levels at sites of release are severely reduced in
fat-3 mutants. All images were taken from the same 50 μm portion of the dorsal nerve cord (in black in the schematic on panel F). Anterior, left; dorsal, up. The nerve cord is shown in red. (A) Colocalization of GFP::UNC-26 with the presynaptic marker SYD-2::RFP in
unc-26-synaptojanin animals. (B and C) Effect of the LC-PUFA AA on synaptojanin localization at release sites. (B) In
unc-26-synaptojanin animals, which have a fully functional FAT-3 protein, GFP::UNC-26 is localized along the dorsal nerve cord and accumulates in synaptic varicosities exhibiting a punctate pattern. In
fat-3unc-26-synaptojanin mutants, which have a nonfunctional FAT-3 protein, GFP::UNC-26 fluorescence is reduced at sites of release and often even puncta are hardly visible. Exogenous AA restores the WT punctate pattern in
fat-3unc-26-synaptojanin mutants. (C) Quantification of the effect of AA on GFP::UNC-26 puncta density. (D) Quantification of GFP::UNC-26 fluorescence at release sites. (E) Quantitative RT-PCR analysis of
unc-26-synaptojanin mRNA levels. Two different control genes,
nhr-23 and β-actin, were used to normalize for the levels of
unc-26-synaptojanin mRNA. No significant difference was detected in the mRNA levels of
fat-3 mutants and
unc-57-endophilin mutants compared with WT animals. Values are represented as the ratio between the mRNA copy number of
unc-26-synaptojanin and that of either β-actin or
nhr-23, and are normalized to WT. (F) UNC-57:: GFP and AMPH-1::GFP localizations appear normal in
fat-3 mutants. In A, B, and F the fluorescence detected outside the nerve cord is gut autofluorescence. In C, D, and E data were plotted as mean ± SEM. Scale bars, 10 μm. **p = 0.026; ***p < 0.001. Dnc, dorsal nerve cord. Alleles:
fat-3(
wa22),
unc-57(
ok310) and
unc-26(
s1710).