Fig. 2

Local stimulation of optoRET in cultured neurons. a Schematic diagram of a neuron, highlighting retrograde signal transmission from a distal, local region (R1) to the cell body (R2). The R1 constitutes a local photoactivated area, indicated by the dashed circle, and R2 is where the activities of AKT and ERK biosensors are monitored. b Representative confocal images of R1 and R2 in locally photoactivated neurons expressing optoRET and biosensors of AKT or ERK activity before and after photoactivation. The illuminated areas are indicated by dashed blue circles. Scale bars = 5 µm. c Quantification of AKT and ERK activation in the cell bodies of neurons by distal, local optoRET stimulations (0–15 min). Data are presented as means ± SEM (AKT in blue, n = 7; ERK in red, n = 5). d Representative confocal images of a neuron expressing optoRET and an F-actin biosensor (LifeAct). The cropped area (dashed red rectangle) is highlighted in time-lapse images on the left, and the photoactivated area is indicated by the dashed blue circle in the image at 0 min. Scale bars = 20 and 10 µm. e Representative time-lapse confocal images of LifeAct, corresponding to the three classes of responses to the local photoactivation of optoRET before (0 min) and after (30 and 60 min) photoactivation. Scale bar = 10 µm. f Comparison of the proportions of responses in non-responding, branching and flower-like structural reorganizations, induced by local photoactivation of optoRET (n = 14) or optoTrkB (n = 8). g Comparison of normalized LifeAct intensities before (0 min) and after (60 min) local photoactivation of optoRET, in the absence (blue, n = 14) or presence (yellow, n = 9) of the Cdc42 inhibitor, ZCL278 (100 µM; 0.5 h pre-incubation). Data are presented as means ± SEM (**p < 0.01; two-way ANOVA); ns, not significant (p > 0.05)