Fig. 1
Development and characterization of optoRET. a Schematic diagram of optoRET. b Representative confocal images of cultured neurons, showing the localization of AKT-PH and EKR-KTR biosensors before and after sustained stimulation by GDNF (50 ng/mL) or light. Scale bars = 5 µm. c, d Graphs showing normalized cytosolic AKT-PH and ERK-KTR cytosol-to-nucleus (C/N) ratios as a function of time. Sustained stimulation was given at 0–40 min by illumination of neurons expressing optoRET (oR), optoRETD387A [oR(D)] or optoTrkB (oB), or by application of GDNF to neurons expressing optoRETfull−length (GDNF). Different numbers of transient light stimulations were also administered to optoRET-expressing neurons, as follows: 1 stimulation at 0 min (× 1), 2 stimulations at 0–2 min (× 2), 6 stimulations at 0–10 min (× 6), and 11 stimulations at 0–20 min (× 11). For each group, n = 4–31 cells. e Comparison of AKT (left) and ERK (right) responsiveness to sustained stimulation with GDNF (AKT: n = 26; ERK: n = 23) optoRET (AKT: n = 31; ERK: n = 36), or optoTrkB (AKT: n = 26; ERK: n = 26). f Comparison of AKT and ERK response size for stimulation with GDNF (AKT: n = 11; ERK: n = 4), optoRET (AKT: n = 20; ERK: n = 28), or optoTrkB (AKT: n = 10; ERK: n = 10). g Comparison of AKT and ERK activation T1/2 following stimulation with GDNF (AKT: n = 6; ERK: n = 4), optoRET (AKT: n = 23; ERK: n = 24), or optoTrkB (AKT: n = 6; ERK: n = 7), obtained by fitting exponential curves. Data are presented as means ± SEM (*p < 0.05, **p < 0.01, ****p < 0.0001; one-way ANOVA); ns, not significant (p > 0.05)