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. 2025 Apr 29;13:RP103757. doi: 10.7554/eLife.103757

Figure 5. Reversible optogenetic ablation of inhibitory synapses with pa-GFE3.

(A) Schematic of paGFE3. Initially, tagRFP-GPHN.FingR-PhoCl2c binds to Gephyrin and PBP-HA-E3 is unbound. After photocleavage with 400 nm light, PBP binds photocleaved PhoCl, recruiting the E3 ligase domain, which ubiquitinates Gephyrin and targets it for degradation by the proteasome. (B) Cultured cortical neuron expressing tagRFP-GPHN.FingR-PhoCl2c + PBP-HA-E3 (pa-GFE3) for 5 days. Closeup of the boxed area shown below. (C) Same neuron as in (B) 5 hr after illumination with 400 nm light for 1 min showing reduced labeling with tagRFP-GPHN.FingR-PhoCl2c. Closeup of the boxed area shown below. (D) Neuron in C immunostained for PBP-HA-E3 (green) and endogenous Gephyrin (red), showing sparse labeling for endogenous Gephyrin. Closeup of the boxed area shown below. (E) Quantification of the total amount of GPHN.FingR and number of GPHN.FingR puncta before and 5 hr after illumination with 400 nm light. ***p < 0.001, Wilcoxon. (F) Cultured cortical neuron expressing pa-GFE3. Closeup of the boxed area shown below. (G) Same neuron as in (F) 5 hr after illumination with 400 nm light for 1 min showing reduced labeling with tagRFP-GPHN.FingR-PhoCl2c. Closeup of the boxed area shown below. (H) Same neuron as in (F), (G) 4 days after illumination showing a recovery of tagRFP-GPHN.FingR-PhoCl2c labeling of synapses. Closeup of the boxed area shown below. (I) Quantification of the total amount of tagRFP-GPHN.FingR-PhoCl2c labeling and number of GPHN.FingR puncta showing recovery of synapses. ***p < 0.001, **p < 0.01, *p < 0.05, ns p > 0.1, Friedman multiple comparison test. Scale bars represent 5 µm.

Figure 5—source data 1. Numerical source data for graphs in Figure 5E, I.

Figure 5.

Figure 5—figure supplement 1. pa-GFE3 has no background activity.

Figure 5—figure supplement 1.

(A) Background activity for the Cryptochrome (Cry2) photoactivation system and PhLIC 5 days after transfection. Cultured neurons transfected with GPHN.FingR-PhoCl+PBP-E3 did not show a significant difference in the intensity of staining with GPHN.FingR-tagRFP compared with those transfected with GPHN.FingR-PhoCl2c only. However, there was a significant reduction in endogenous Gephyrin between cultured neurons transfected with Gephyrin.FingR-Cry2 alone vs. with CIB1-E3. ***p < 0.001, Mann–Whitney; ns, p > 0.05, Mann–Whitney. (B) Cultured neuron expressing transcriptionally regulated tagRFP-Gephyrin.FingR-PhoCl immunostained for tagRFP (red) and endogenous Gephyrin (green). Merge shows colocalization of tagRFP-Gephyrin.FingR-PhoCl with endogenous Gephyrin in yellow. (C) Neuron expressing GPHN.FingR-PhoCl2c alone before exposure to 400 nm light. (D) Same neuron as in (C) after exposure to 400 nm light. There is no apparent loss of labeling. (E) Quantitation of labeling by tagRFP-GPHN.FingR-PhoCl2c in cells transfected with GPHN.FingR-tagRFP alone and then exposed to 400 nm for 1 s and incubated for 5 hr. Staining with GPHN.FingR-tagRFP was compared before and after light exposure. **p < 0.01, ns p > 0.05. (F) Same neuron as in Figure 5F–H showing a comparison of tagRFP-GPHN.FingR-labeled puncta before (green) vs. after (magenta) exposure to 400 nm light.
Figure 5—figure supplement 1—source data 1. Numerical source data for graphs in Figure 5—figure supplement 1A, E.