Fig. 1

M120I and R533* mutants reduce dendritic spine density and mEPSC frequency. a Upper: Schematic of the protein structure of CTTNBP2. Seven human ASD-linked mutations (in parentheses) and their corresponding residues in mouse protein are indicated. Binding partners of each domain are noted below. N, N-terminal region; CC, coiled-coil domain; Mid, middle region; P-rich, proline-rich domain. Middle: Predicted secondary structure of CTTNBP2 by I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER). Red part represents alpha-helix, and black represents coil. Lower: IUpred2A (https://iupred2a.elte.hu) prediction of disordered regions of CTTNBP2. Higher score indicates a higher degree of disorder. b Confirmation of expression of Myc-tagged CTTNBP2 ASD-linked mutant proteins in COS1 cells. β-actin and α-tubulin were used as internal controls. c The effect of CTTNBP2 ASD-linked mutations on dendritic spine density. M120I and R533* mutation showed the most dramatic reduction in dendritic spine density. Representative image of cultured hippocampal neurons that express HA-tagged WT or ASD-linked mutant proteins (viewed in red). Dendritic morphology was outlined by GFP-actin (visualized in green). Quantification results of dendritic spine density are shown. The quantification results of vector, WT and R533* have been reported previously [20]. Since those three groups of experiments were performed at the same time as the experiments on the other mutant proteins targeted for this study, we compare all mutants together here. d M120I and R533* mutants reduce mEPSC frequency but not amplitude. Data represent mean ± SEM. Each dot in (c) and (d) indicates the individual result for a dendritic segment and neuron, respectively. The results were collected from three (c) and ten (d) independent cultures. c Kruskal–Wallis test with Dunn’s multiple comparison test were used to compare mutant protein with the wild-type (WT) group (*) or vector control (#). d One-way ANOVA was used for analysis. See Additional file 2: Table S1 for all statistical data and exact sample size for each group. * or #P < 0.05; **P < 0.01; *** or ###P < 0.001. Scale bar, 5 μm