Jip3 interaction with JNK was necessary for pJNK clearance and the prevention of axon swellings.

<p>(A–C) Axon terminal swellings and pJNK accumulation were rescued by Jip3 but not by Jip3ΔJNK. Axonal swellings were visualized live by <i>neurod:EGFP</i> transgene expression; following live imaging, pJNK was assayed by individual larva immunolabeling at 4 dpf. White arrowheads mark axon terminals expressing the DNA constructs. Yellow arrow points to a swelling in an axon not expressing Jip3-mCherry. Red arrowhead denotes an underlying pJNK positive cell not expressing Jip3-mCherry. (D) Ratio of axon terminal swellings in each class (mild = small swellings, severe = large swellings) show rescue of axonal swellings by full-length Jip3 but not Jip3ΔJNK. <i>DNA</i> indicates the rescue construct injected; Jip3 = full-length Jip3-mCherry; ΔJNK = Jip3ΔJNK-mCherry; Con = uninjected control. Embryo genotype, determined by control axon terminal morphology, is indicated below each bar. (E) Ratio of pJNK levels in Jip3 or Jip3ΔJNK expressing axon terminals to those not expressing the rescue construct at 4 dpf. Jip3, but not Jip3ΔJNK, suppressed increased pJNK levels in <i>jip3^nl7</i> (Wilcoxon rank-sum; *-<i>p</i><0.01). (F) Induction of constitutively active JNK3 tagged with EGFP (caJNK3; green) for 15 hours at 4 dpf increased the level of pJNK immunofluorescence concomitant with the induction of swellings shown by both the caJNK3-EGFP fill and Tag1 immunolabeling of neuronal membranes. Arrowhead points to a caJNK3-EGFP expressing axon. Yellow arrow indicates an axon terminal in the same NM not expressing this construct. (G) Axon terminal swellings were absent in axon terminals expressing an inactive form of the same construct (caJNK3-IA), indicating that JNK activation was necessary to induce swellings. (H,I) Efficacy of both caJNK3 and caJNK3-IA were assayed by Western blot analysis of phospho-cJun, a downstream target of active JNK. While whole embryo overexpression of caJNK3-EGFP by RNA injection induced elevated levels of phospho-cJun at 24 hpf (H), similar expression of the inactive form of caJNK3-EGFP (caJNK3-IA) failed to induce a similar increase (I). Scale bars = 10 µm.