Fig 2: Overexpressing Shank3 phospho-mutants does not change mEPSC frequency at baseline or during the scaling protocols.Quantification of average mEPSC frequency in neurons pooled from the dataset in Figure 6 (WT, n = 16, WT + TTX, n = 9, WT + BIC, n = 8, DD, n = 7, DD + TTX, n = 11, AA, n = 9, AA + BIC, n = 14; Kruskal- Wallis test: p = 0.2483).

Fig 3: Overexpressing wild-type Shank3 does not impact synaptic surface GluA2 intensity at baseline, or the magnitude of scaling up.Quantification of average synaptic sGluA3 intensity in neurons overexpressing Shank3-WT or a control GFP vector with and without TTX treatment (EV UN, n = 40, EV TTX, n = 45, WT UN, n = 33, WT TTX, n = 34). EV, empty vector; WT, Shank3-WT; UN, untreated.

Fig 4: Contribution of the Shank3 N-terminus to the postsynaptic targeting of δ-catenin. a A new Shank3 construct lacking the N-terminal SPN-Ank domain was used for transfection of primary rat hippocampal neurons. The transfected neurons were fixed and stained for MAP2 and PSD-95. The green signal of Shank3 shows a high colocalization rate with PSD-95 indicating that the synaptic localization of Shank3 is not dependent on N-terminal domain (scale bar for overview images: 20 µm and for magnified images: 5 μm). b RFP-δ-catenin was coexpressed with either an empty pHAGE-GFP construct as a control or with different Shank3 (WT, L68P or ΔN-terminal) constructs. The neurons were fixed and stained for MAP2 as dendritic marker and PSD-95 as synaptic marker (scale bar 5 µm). c Quantitative analysis of δ-catenin signal intensity in 100 spines of 10 neurons obtained from two independent transfection, showed L68P variant of Shank3 increases the δ-catenin level in dendritic spines compared to Shank3 WT, whereas ΔN-terminal Shank3 is unable of changing localization of δ-catenin in neurons and shows a similar effect as pHAGE-GFP control construct. The results show that Shank3 N-terminus is responsible for recruiting δ-catenin to the postsynaptic sites (n = 10, one-way ANOVA with Dunnett’s Test, **p < 0.01, ****p < 0.0001, mean ± SD)

Fig 5: Changes in the phosphorylation state of Shank3 are crucial for bidirectional synaptic scaling.(A, B) Representative miniature excitatory postsynaptic current (mEPSC) recordings from neurons overexpressing Shank3 WT (A) or DD mutant (B) during scaling up. (C) Quantification of average mEPSC amplitude in (A) (WT, n = 8, WT + tetrodotoxin [TTX], n = 9; unpaired two-tailed t-test: **p=0.0074). (D) Quantification of average mEPSC amplitude in (B) (number of neurons: DD, n = 12, DD + TTX, n = 14; unpaired two-tailed t-test: p=0.5708). (E, F) Representative traces of mEPSCs recorded from neurons overexpressing Shank3 WT (E) or AA mutant (F) during scaling down. (G) Quantification of average mEPSC amplitude in (E) (number of neurons: WT, n = 8, WT + bicuculline [BIC], n = 8; Mann–Whitney test: *p=0.0148). (H) Quantification of average mEPSC amplitude in (F) (AA, n = 9, AA + BIC, n = 14; unpaired two-tailed t-test: p=0.8612). Also see Figure 6—figure supplement 1, Figure 6—figure supplement 2, and Figure 6—source data 1. Figure 6—source data 1.Source data for Figure 6.