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. 2018 Aug 3;12:217. doi: 10.3389/fncel.2018.00217

Figure 13.

Figure 13

Characterization of protein localization, dendritic spine density and morphology in DIV14-15 hippocampal neurons expressing EGFP and mCherry (control), wild-type SrGAP3-mCherry or mutated SrGAP3-E469K-mCherry. (A) Primary structure of human SrGAP3 with mutation E469K indicated. (B) Representative maximum projections of confocal Z-stacks for EGFP, mCherry, and wild-type and mutated SrGAP3 localization. Scale bars 4 μm. (C) The quantification of protein localization revealed similar localization of both wt and mutated SrGAP3 to dendritic spines. Spine-to-dendrite fluorescence intensity ratios: control: 0.99 (n = 34 neurons, 337 spines), SrGAP3: 1.32 (n = 25 neurons, 257 spines), SrGAP3-E469K: 1.42 (n = 23 neurons, 229 spines). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; crosses represent sample means. **p < 0.01, ***p < 0.001 as determined by one-way ANOVA test with Games-Howell post-hoc test. (D) Maximum projections of confocal Z-stacks of dendrites from neurons overexpressing EGFP along with either mCherry, SrGAP3-mCherry or SrGAP3-E469K-mCherry. Only EGFP channel is shown and was used to assess spine density and morphology. Scale bar, 4 μm. (E) Quantification of dendritic spine density calculated as number of spines per 1 μm of dendrite. The first left cluster of bars represents total spine density. Densities of thin, mushroom, stubby, and total spines were as follows: wt: thin = 0.23, mushroom = 0.16, stubby = 0.06, total = 0.45 spines/μm (n = 34 neurons, 1,459 spines, 3,401 μm dendrite); SrGAP3: thin = 0.29, mushroom = 0.19, stubby = 0.07, total = 0.54 spines/μm (n = 25 neurons, 1,431 spines, 2,792 μm dendrite); SrGAP3-E469K: thin = 0.28, mushroom = 0.19, stubby = 0.07, total = 0.54 spines/μm (n = 23 neurons, 1,087 spines, 2,025 μm dendrite). Data is pooled from 3 experiments and represented as mean ±SEM. *p < 0.05 as determined by one-way ANOVA test with Games-Howell post-hoc test. (F) Cumulative distributions of the width, length and the ratio of width to length of dendritic spines for neurons expressing either mCherry (control), SrGAP3-mCherry or SrGAP3-E469K-mCherry. Curves are a combination of data points each representing an individual spine. Matching tail regions of the curves are not shown. Significant differences were not detected by pairwise two-sample Kolmogorov-Smirnov test.