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. 2020 Mar 4;40(10):2000–2014. doi: 10.1523/JNEUROSCI.0893-19.2020

Figure 5.

Figure 5.

Shank3 949RRK951 to AAA mutation disrupts colocalization of activated CaMKIIα. A, Immunoblots of undifferentiated, differentiated, and transfected/differentiated STHdh+/+ cells, with a WT mouse forebrain lysate as positive control. Shank3 and CaMKIIα are not expressed in nontransfected STHdh+/+ cells, and transfected mApple-tagged CaMKIIα (mAp-CaMKIIα) is T286-phosphorylated. B, Representative images of differentiated STHdh+/+ cells expressing GFP-Shank3-WT with mAp-CaMKIIα-WT (left), mAp-CaMKIIα-T286D (middle), or mAp-CaMKIIα-T286A (right). Inset, Regions (dashed line box) of the processes containing punctate GFP signals (arrowheads) that overlap with mApple signal from CaMKIIα-WT and -T286D, but not -T286A. C, Intensity correlation analysis quantifying the colocalization of GFP and mAp signals in transfected and differentiated STHdh+/+ cells in B. Each data point represents an ICQ value from a single cell, with 7–12 cells analyzed from each of three independent cultures/transfections. ICQ values for GFP-Shank3-WT and either mAp-CaMKIIα-WT (mean ± SEM: 0.29 ± 0.02) or mAp-CaMKIIα-T286D (0.36 ± 0.02) were significantly more colocalized compared with mAp-CaMKIIα-T286A (0.17 ± 0.02) (one-way ANOVA, F(2,71) = 21.35, p < 0.0001). Tukey's post hoc test: ***p < 0.001, ****p < 0.0001. D, Representative images of differentiated STHdh+/+ expressing GFP-Shank3-WT or GFP-Shank3-AAA with soluble mAp or mAp-CaMKIIα-WT. Inset, Expanded regions of the processes, as in B. E, Intensity correlation analysis quantifying the colocalization of GFP and mAp signals in transfected and differentiated STHdh+/+ cells in C. GFP-Shank3-WT and mAp-CaMKIIα-WT (0.31 ± 0.02) are significantly more colocalized than GFP-Shank3-WT and mAp (0.07 ± 0.01) or GFP-Shank3-AAA and mAp-CaMKIIα (0.09 ± 0.02) (one-way ANOVA, F(2,70) = 39.55, p < 0.0001). Tukey's post hoc test: ****p < 0.0001. Scale bars: B, D, 2.5 μm.