Figure S5.
Calmodulin-dependent increase in the number of endophilin A1 puncta correlates with spine size during the acute phase of sLTP. (A) Cultured hippocampal neurons transfected with pLL3.7-DsRed on DIV12 were pretreated with DMSO or MK801 for 30 min before cLTP induction with glycine for 5 min on DIV16, immunostained for EEN1 (pseudocolored green) and PSD95 (pseudocolored red), and imaged by 3D-SIM. Shown are representative images with DsRed pseudocolored blue. Spines were outlined manually. Scale bars represent 4 µm in upper panels and 500 nm in magnified images. (B) Quantification of spine size in A. (C) Quantification of the number of EEN1 puncta in spines in A. (D) Quantification of the area of EEN1 or PSD95 puncta in spines in A. Data are expressed as mean ± SEM or plotted along with mean ± SEM for each group in C and D (n = 13, n = 570 for − Gly; n = 13, n = 502 for + Gly; n = 14, n = 543 for + MK801 + Gly). P values were calculated using one-way ANOVA by Dunnett post hoc test. ***, P < 0.001 when compared with − Gly. (E and F) Scatterplot of the number or area of EEN1 puncta versus the size of spine head with linear fits using linear regression analysis. (G) Scatterplot of the number of EEN1 puncta versus the area of PSD95 puncta in spines with linear fits using linear regression analysis. In E–G, n = 13, n = 570 for − Gly; n = 13, n = 502 for + Gly; n = 14, n = 543 for + MK801 + Gly. (H) Effect of W-7 or KN-62 on EEN1 puncta in spines. Neurons were pretreated with DMSO, W-7 or KN-62, before cLTP induction with glycine for 5 min on DIV16, immunostained and imaged by 3D-SIM. Shown are representative images. Scale bars represent 4 µm in upper panels and 500 nm in magnified images. (I) Quantification of spine size in H. (J) Quantification of the number of EEN1 puncta in spines in H. Data are expressed as mean ± SEM or plotted along with mean ± SEM for each group in I and J (n = 14, n = 577 for − Gly; n = 14, n = 596 for + Gly; n = 14, n = 588 for + W-7 + Gly; n = 14, n = 540 for + KN-62 + Gly). P values were calculated using one-way ANOVA by Dunnett post hoc test. ***, P < 0.001 when compared with − Gly.

Calmodulin-dependent increase in the number of endophilin A1 puncta correlates with spine size during the acute phase of sLTP. (A) Cultured hippocampal neurons transfected with pLL3.7-DsRed on DIV12 were pretreated with DMSO or MK801 for 30 min before cLTP induction with glycine for 5 min on DIV16, immunostained for EEN1 (pseudocolored green) and PSD95 (pseudocolored red), and imaged by 3D-SIM. Shown are representative images with DsRed pseudocolored blue. Spines were outlined manually. Scale bars represent 4 µm in upper panels and 500 nm in magnified images. (B) Quantification of spine size in A. (C) Quantification of the number of EEN1 puncta in spines in A. (D) Quantification of the area of EEN1 or PSD95 puncta in spines in A. Data are expressed as mean ± SEM or plotted along with mean ± SEM for each group in C and D (n = 13, n = 570 for − Gly; n = 13, n = 502 for + Gly; n = 14, n = 543 for + MK801 + Gly). P values were calculated using one-way ANOVA by Dunnett post hoc test. ***, P < 0.001 when compared with − Gly. (E and F) Scatterplot of the number or area of EEN1 puncta versus the size of spine head with linear fits using linear regression analysis. (G) Scatterplot of the number of EEN1 puncta versus the area of PSD95 puncta in spines with linear fits using linear regression analysis. In E–G, n = 13, n = 570 for − Gly; n = 13, n = 502 for + Gly; n = 14, n = 543 for + MK801 + Gly. (H) Effect of W-7 or KN-62 on EEN1 puncta in spines. Neurons were pretreated with DMSO, W-7 or KN-62, before cLTP induction with glycine for 5 min on DIV16, immunostained and imaged by 3D-SIM. Shown are representative images. Scale bars represent 4 µm in upper panels and 500 nm in magnified images. (I) Quantification of spine size in H. (J) Quantification of the number of EEN1 puncta in spines in H. Data are expressed as mean ± SEM or plotted along with mean ± SEM for each group in I and J (n = 14, n = 577 for − Gly; n = 14, n = 596 for + Gly; n = 14, n = 588 for + W-7 + Gly; n = 14, n = 540 for + KN-62 + Gly). P values were calculated using one-way ANOVA by Dunnett post hoc test. ***, P < 0.001 when compared with − Gly.

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