Figures and data
Mdm2.RING ubiquitinates PSD-95.
A) Comparison of cell lysate from COS7 cells transfected with PSD-95-myc alone, PSD-95-myc + Dox-induced TRE-PSD-95.FingR-RINGMDM2, and PSD-95-myc + PSD-95.FingR-RING MDM2 + TAK243, a ubiquitination inhibitor. Cells expressing TRE-PSD-95.FingR-RINGMDM2 were treated with 1μg/ml Doxycycline for 4 hr to induce expression of PSD-95.FingR-RING and showed a reduction in PSD-95-myc. Cells treated with 20 μM TAK243 showed no significant reduction in PSD-95-myc. * p < 0.05, ns p > 0.6 Quantitation showed a significant reduction in PSD-95 expression when co-expressed with PSD-95.FingR-RINGMDM2 in COS7 cells, but not when PSD-95 is expressed alone or when PSD-95 and PSD-95.Fn-RING MDM2 are co-expressed with 20 μM TAK243. * p < 0.05, ANOVA with multiple comparisons. ns, p > 0.05.
B) Cultured cortical neuron expressing transcriptionally regulated PSD-95.FingR-tagRFP before induction of PSD-95.FingR-HA-RINGMdm2 expression with Dox.
C) Same neuron as in C) after induction of PSD-95.FingR-HA-RINGMDM2 expression with Dox shows a reduction in PSD-95.FingR-tagRFP labeling.
D) Immunostaining of the neuron in D) for PSD-95.FingR-HA-RINGMDM2 (green) and endogenous PSD-95 (red).
E) Quantification of the number of PSD-95 puncta and the total amount of PSD-95 labeled by PSD-95.FingR-tagRFP before and after expression of PSD-95.FingR-HA-RINGMDM2. ** p < 0.01
Error bars represent ± sem Scale bars 5 µm.
PFE3 reversibly ablates PSD-95 in neurons.
A) Cultured cortical neuron expressing PSD-95.FingR-tagRFP before induction of PFE3. Closeup of the boxed area shown below.
B) Same neuron as in A) after expression of PFE3 for 48 hr shows a dramatic reduction in PSD-95.FingR labeling. Closeup of the boxed area shown below confirms the lack of punctate labeling by PSD-95.FingR-tagRFP.
C) Neuron in B) immunostained for PFE3 (green) and endogenous PSD-95 (red) confirms the lack of punctate labeling by PSD-95.FingR. Closeup of the boxed area shown below.
D) Quantifications of the total amount of PSD-95.FingR labeling and the # of PSD-95 puncta show significant reductions following expression of PFE3. *** p = 0.001, Wilcoxon.
E-G) Cultured cortical neurons immunostained for endogenous PSD-95 in red and GluA1 in green. E) Untransfected. F) Following RandE3 (blue) expression for 48 hr, GluA1, and PSD-95 staining is intact. G) Following PFE3 expression (blue) for 48 hr, GluA1 and PSD-95 expression is markedly diminished.
H) Quantification of the percentage of PSD-95 puncta positive for GluA1 staining. ns p > 0.05, *** p < 0.0001, Mann Whitney.
I) Cultured neuron expressing PSD-95.FingR-tagRFP.
J) Same neuron as in I) 48 hr after induction of PFE3 expression shows a dramatic reduction in labeling with PSD-95.FingR-tagRFP.
K) Same neuron as in I), J) showing recovery of synapses five days after removal of PFE3.
L) Quantification of the total amount of PSD-95.FingR-tagRFP labeling and the # of PSD-95.FingR-tagRFP-labeled puncta. ** p < 0.01, * p < 0.05, ns p > 0.05
Scale bars 5 µm.
Expression of PFE3 reduces synaptic transmission in vivo.
A) Schematic depiction of the retinal circuit used to assess AMPA receptor function. Channelrhodopsin-2 is expressed in type 6 cone bipolar cells (CBC6), which are presynaptic to the On α-retinal ganglion cell (α-RGC). Synaptic output of photoreceptors is pharmacologically blocked.
B) Population data comparing peak EPSC amplitude for retinas from mice infected with PFE3 and control viruses. Responses were generated with a 495 nm, 500 ms flash of light. Symbols are values for individual cells. Statistical comparison was obtained using the Wilcoxon signed-rank test. Error bars represent ± s.e.m.
C)-D) Sample whole cell patch clamp recording from an On α-RGC infected with the PFE3 virus (C) or a RandE3 control virus (D).
Testing of PhLIC, a photoactivatable dimer.
A) Schematic of PhLIC (PhoCl-based Light-Inducible Complex). Full-length PhoCl is photocleaved with 400 nm light, exposing an epitope recognized by the PhoCl Binding Peptide (PBP), leading to dimerization of photocleaved PhoCl and PBP.
B) COS7 lysate expressing PBP-HA-GST was incubated with purified full-length or photocleaved Myc-PhoCl2c. PBP-HA-GST pulls down photocleaved but not full-length PhoCl2c.
C) Schematic of Golgi-targeting assay. COS7 cell expressing Golgi-targeted PhoCl2c in green and PBP-tagRFP in red before (left) and 3 hr after illumination with 400 nm light for 10 sec every 30 sec for 3 min (right).
D) Quantification of the ratio of the total fluorescence associated with PBP-tagRFP localized in the cytosol (Icytosol) vs. at the Golgi (IGolgi) before and 3 hr after photocleaving PhoCl2c. *** p < 0.001
E) COS7 cell before photoactivation showing PBP-tagRFP (left) and a merge (right) of PBP-tagRFP (red) and PhoCl2c-GTS (green).
F) Same COS7 cell as in E) following photoactivation with 400 nm light for 1 min showing PBP-tagRFP (left) and a merge (right) of PBP-tagRFP (red) and PhoCl2c (green). Scale bar represents 5 µm.
Reversible optogenetic ablation of inhibitory synapses with pa-GFE3
A) Schematic of paGFE3. tagRFP-GPHN.FingR-PhoCl2c binds to Gephyrin. 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 < 0.05, ns p > 0.1, Friedman test multiple comparisons.
Scale bars represent 5 µm.
chGFE3 reversibly ablates Gephyrin
A) Schematic of chGFE3. The addition of TMP-HaloTag ligand (TH) dimerizes HaloTag and eDHFR, leading to the recruitment of the E3 ligase to Gephyrin, which ubiquitinates and degrades it.
B) Cultured cortical neuron expressing GPHN.FingR-HaloTag and eDHFR-RINGXIAP (chGFE3) for 4 days. Closeup of the boxed area shown below.
C) Same neuron as in B) 4 hr after addition of 100 nM TH. Closeup of the boxed area shown below.
D) Immunocytochemistry of the neuron in C) for endogenous Gephyrin (red) and eDHFR-E3XIAP (green). Note the lack of red puncta on dendrites labeled green. Closeup of the boxed area shown below.
E) Quantification of total Gephyrin labeled by GPHN.FingR-tagRFP and number of GPHN.FingR puncta after 4 hr of incubation with 100 nM TH. *** p < 0.001, Wilcoxon.
F) Cultured cortical neuron expressing GPHN.FingR-HaloTag and eDHFR-RING for 4 days. Closeup of the boxed area shown below.
G) Same neuron as in F) after 24 hr incubation with 100 nm TH showing loss of Gephyrin. Closeup of the boxed area shown below.
H) Neuron in G) immunostained for endogenous Gephyrin (red) and DHFR-E3 (green). Closeup of the boxed area shown below.
I) Quantification of the total amount of Gephyrin labeling by the GPHN.FingR after the addition of TH. **** p < 0.0001, Friedman Test multiple comparisons.
J) Schematic illustrating dissociation of chGFE3 with the addition of TMP.
K) Cultured cortical neuron expressing GPHN.FingR-HaloTag and eDHFR-RINGXIAP for 4 days. Closeup of the boxed area shown below.
L) Same neuron as in K) after 24 hr incubation with 100 nm TH showing loss of Gephyrin. Closeup of the boxed area shown below.
M) Same neuron as in L) after 48 hr incubation with 100 µM TMP showing recovery of Gephyrin puncta. Closeup of the boxed area shown below.
N) Quantification of the total amount of Gephyrin labeling by the GPHN.FingR after the addition of TH and then TMP. **** p < 0.0001, * p < 0.05, ns p > 0.05 Friedman Test multiple comparisons.
Scale bar represents 5 μm.
