Figure 8. Resonance and critical activation periods during WTS-evoked JNK signalling.

(a) Current understanding suggests cerebellar neuron WTS-evoked c-Jun involves nuclear pool of JNK, whereas cytoplasmic JNK was thought not to impact c-Jun regulation. However, data presented in this figure suggest that specific periodicities of cytoplasmic JNK inhibition does in fact lead to regulation of c-Jun transactivation (dotted red arrow). (b) Neurons expressing GAL4-cJun reporter (Figs 6 and 7) without ΔMEKK1 exhibit baseline GAL4-cJun signal without WTS (not phosphorylation-dependent because mutation of the sites has little effect³¹). Four hours WTS (20 h post-transfection) induces ∼twofold activation. In darkness, no targeted OptoJNKi had any effect. Constitutive JBD targeted to cytoplasm (NES) had no effect but NLS-constitutive JBD reduced the activation >50% as in previous reports²³ (n=8). (c) Neurons as in Fig. 8b were illuminated as indicated (1.5 s pulses). H2B-OptoJNKi (targeted to chromatin) evoked strongest inhibition, followed by NLS-OptoJNKi. NES-OptoJNKi had no effect at 7.5 s periodicity but significantly suppressed at 3 min periodicity (n=8). (d,e) Neurons were transfected as in Fig. 8c showed increasing NES-OptoJNKi-dependent inhibition of 4 h WTS-evoked GAL4-cJun response over illumination periodicities from 7.5 to 600 s at which point inhibition was maximal. At 1,800 s periodicity, no inhibition was detected. (e) Percentage inhibition compared with the data normalization condition (CM/darkness/no OptoJNKi) from the experiments in Fig. 8d is shown and significance of differences from control are highlighted (n=4, or 6 for CM samples in darkness). (f,g) Neurons were illuminated by constant 1.5 s pulse per 7.5 s periodicity, either for the full 4 h, or only during the first two, second two or third two hours of a 4 h WTS period. (g) Percentage inhibition compared with the data normalization condition (CM/darkness/no OptoJNKi) from the experiments of Fig. 8f is shown. Statistical analysis indicates that inhibition was only obtained when illumination took place during the first hour and NLS-OptoJNKi was present (n=3). Mean±s.e.m. is shown; ns indicates not significant, *P<0.05, **P<0.01, ***P<0.001. Analysis was carried out using by two-way ANOVA/Bonferroni and Neuman–Keuls post-tests (Supplementary Data 1).