Fig. 7: Minimal illumination conditions sufficient to inhibit the JNK pathway inhibition with mTq2-optoJNKi and demonstrate fluorescent tag-dependent sensitivity of actuation.

a To evaluate activation of the JNK pathway in real-time, multipoint epi-fluorescence microscopy of neurons expressing JNK reporter was carried out during trans-illumination of selected wells by blue LEDs gated by microscope imaging acquisition. b Based on parameters from Table 1, and a 1 s 0.06 mW/cm² flash of blue light every 7.5 s, the % of construct in adduct state stabilises after about 1 min as shown. The mTq2 construct reaches approximately fivefold greater proportion in adduct state compared with the Ypet construct, whereas the mScarlet construct exhibits minimal adduct state under these conditions. c–f Changes in cytoplasm:nuclear ratios of the JNK pathway reporter JNKktr (increased translocation from the nucleus indicates activation of JNK) are shown in response to treatment with JNK-activator anisomycin. Cultured cortical neurons were infected with AAVs encoding synapsin-promoter driven JNKktr fused to miRFP670 and optoJNKi fused to either mTq2, Ypet or mScarlet as shown. The light-on samples (turquoise datapoints) were exposed to light as shown in a. The mTq2-optoJNKi expressing neurons exhibited significant light-dependent inhibition, the Ypet-optoJNKi had a barely detectable and not significant effect in response to light whereas the mScarlet-optoJNKi expressing cells did not respond to light. This is consistent with the predictions shown in b. Means ± SEM (n = 4 wells) are shown. ***P < 0.001, ns denotes non-significance, by two-way ANOVA. Source data are provided as a Source Data file.