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. 2020 Oct 6;9:e57395. doi: 10.7554/eLife.57395

Figure 2. Activation of optoRaf but not optoAKT increases C4da neuron dendrite complexity.

(A–D) 15 min blue light stimulation activates optoRaf and optoAKT in flies in vivo. After the light was off, the downstream effectors inactivated gradually. (A) The body walls from WT and optoRaf expressing larvae were dissected and stained for pERK1/2. The 15 min continuous light illumination leads to the enhanced fluorescent intensity and nuclear translocation of pERK in the optoRaf-expressing C4da neurons (labeled by ppk-CD4tdGFP). pERK signal is significantly increased even at 45 min after the light was off. Notably, the ERK signaling is not activated by light stimulation in optoAKT-expressing neurons. C4da neuron cell bodies are outlined by dashed white lines. Scale Bar = 10 μm. (B) Phospho-p70S6K is activated by light illumination in optoAKT expressing neurons, and gradually returned to baseline after blue light was shut off. (C) Qualification of pERK fluorescence intensity in (A). The intensity of pERK in transgenic larvae was normalized to that of WT. WT (0 min off) N = 19, optoRaf (0 min off) N = 16, optoRaf (15 min off) N = 19, optoRaf (45 min off) N = 18, optoRaf (dark) N = 18, optoAKT (0 min off) N = 19 neurons. (D) Qualification of phospho-p70S6K fluorescence intensity in (B). The intensity of phospho-p70S6K in transgenic larvae was normalized to that of WT. WT (0 min off) N = 18, optoAKT (0 min off) N = 19, optoAKT (15 min off) N = 23, optoAKT (45 min off) N = 20, optoAKT (dark) N = 16, optoRaf (0 min off) N = 23 neurons. (E–G) Activation of Raf/MEK/ERK but not AKT signaling by 72 hr' light stimulation increases dendrite outgrowth and branching in C4da neurons. (E) Representative images of C4da neurons from WT, optoRaf and optoAKT expressing larvae with 72 hr' light stimulation and the unstimulated controls. Neurons were reconstructed with Neuronstudio. Scale bar = 50 μm. (F) Quantification of total dendrite length of C4da neurons. (G) Qualification of dendritic branch number. WT (light) N = 21, optoRaf (light) N = 21, optoRaf (dark) N = 21, optoAKT (light) N = 20, optoAKT (dark) N = 20 neurons. All data are mean ± SEM. The data were analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test, **p<0.01, ***p<0.001. See also Figure 3—figure supplements 1–2.

Figure 2—source data 1. Activation of optoRaf but not optoAKT increases C4da neuron dendrite complexity.

Figure 2.

Figure 2—figure supplement 1. Activation kinetics of optoRaf and optoAKT in fly sensory neurons.

Figure 2—figure supplement 1.

(A–B) 5 min light illumination is sufficient to activate optoRaf, while longer light stimulation can further increase pERK intensity and induce ERK nuclear translocation. Blue light is applied for 0, 5, 10, and 15 min to optoRaf expressing larvae. The fluorescence intensity of pERK is normalized to that of neurons with no blue light treatment (0 min). optoRaf (0 min) N = 20, optoRaf (5 min) N = 21, optoRaf (10 min) N = 18, optoRaf (15 min) N = 16. (C–D) The intensity of phospho-p70S6K is significantly increased after 10 min light illumination in optoAKT expressing neurons. Blue light is applied for 0, 5, 10, and 15 min to optoAKT expressing larvae. The fluorescence intensity of phospho-p70S6K is normalized to that of neurons with no blue light treatment (0 min). optoAKT (0 min) N = 17, optoAKT (5 min) N = 18, optoAKT (10 min) N = 17, optoAKT (15 min) N = 17. Data are mean ± SEM, analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. **p<0.01, ***p<0.001.
Figure 2—figure supplement 1—source data 1. Activation kinetics of optoRaf and optoAKT in fly sensory neurons.
Figure 2—figure supplement 2. The specific activation of ERK/p70S6K in C4da neurons.

Figure 2—figure supplement 2.

Downstream effectors are specifically activated in optoRaf or optoAKT expressing neurons by light illumination. The arrowheads mark da neurons and arrows mark epithelial cells. Scale S6K = 10 μm.
Figure 2—figure supplement 3. Inactivation kinetics of optoRaf, and activation of optoRaf does not upregulate phospho-p70S6K.

Figure 2—figure supplement 3.

Phospho-p70S6K was activated by 15 min light illumination in optoAKT expressing neurons, and inactivated 15 min after the light was off. No enhancement of phospho-p70S6K fluorescent intensity was observed in optoRaf-expressing neurons with light stimulation.