IRSp53–actin interaction regulates dendritic spine morphology and modulates synaptic enrichment of IRSp53. (A) Representative fluorescence images of cultured IRSp53 knockout cortical neurons with the re-expression of mEGFP-tagged IRSp53 WT or mutant constructs at DIV7. mCherry was cotransfected as the cell fill. Endogenous IRSp53 was deleted upon Cre expression, and mEGFP-tagged IRSp53 WT or mutant cDNA was transfected for 10 d. At DIV17, live imaging was performed without fixation. (B–D) Quantification of the imaging data showing spine enrichment (B) measured using Image J and spine head width (C) and spine density (D) measured using IMARIS. 24–43 neurons from three independent batches of cultures were imaged for each group in double-blinded mode (n = 43 for mCherry, n = 42 for Cre only, n = 36 for Cre coexpression with IRSp53 WT, n = 26 for Cre coexpression with IRSp53_K4E, and n = 24 for IRSp53_RKE). It is noted that data in Fig. 3 H were collected from the same set of experiments with the same control groups (i.e., Cre only and Cre co-expression with IRSp53 WT). Error bars indicate mean ± SEM. ***, P < 0.001, ****, P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test.