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

Figure 2.

Figure 2

Characterization of protein localization, dendritic spine density and morphology in DIV14-15 hippocampal neurons expressing EGFP and mCherry (control), wild-type α-actinin-4-mCherry or mutated α-actinin-4-M554V-mCherry. (A) Representative maximum projections of confocal Z-stacks for EGFP, mCherry, and wild-type and mutated α-actinin-4 localization. Scale bars, 4 μm. (B) The quantification of protein localization revealed enrichment of wt α-actinin-4 to dendritic spines and the decrease in accumulation for α-actinin-4-M554V. Spine-to-dendrite fluorescence intensity ratios: control: 0.98 (n = 34 cells, 339 spines), α-actinin-4: 6.64 (n = 26 cells, 260 spines), α-actinin-4-M554V: 4.1 (n = 30 cells, 299 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 < 0.001 as determined by one-way ANOVA test with Games-Howell post-hoc test. (C) Maximum projections of confocal Z-stacks of dendrites from neurons overexpressing EGFP along with either mCherry, α-actinin-4-mCherry or α-actinin-4-M554V-mCherry. Only EGFP channel is shown and was used to assess spine density and morphology. Scale bar, 4 μm. (D) 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 = 0.25, mushroom = 0.15, stubby = 0.07, total = 0.48 spines/μm (n = 34 neurons, 1,937 spines, 4,091 μm dendrite); α-actinin-4: thin = 0.18, mushroom = 0.21, stubby = 0.08, total = 0.46 spines/μm (n = 26 neurons, 1,229 spines, 2,816 μm dendrite); α-actinin-4-M554V: thin = 0.24, mushroom = 0.18, stubby = 0.09, total = 0.51 spines/μm (n = 30 neurons, 1,750 spines, 3,573 μm dendrite). Data is pooled from 4 experiments and represented as mean ± SEM. *p < 0.05, **p < 0.01 as determined by one-way ANOVA test with Bonferroni post-hoc test. (E) Cumulative distributions of the width, length and the ratio of width to length of dendritic spines for neurons expressing either mCherry (control), α-actinin-4-mCherry or α-actinin-4-M554V-mCherry. Curves are a combination of data points each representing an individual spine. Matching tail regions of the curves are not shown. *p < 0.05, ***p < 0.001 as determined by pairwise two-sample Kolmogorov-Smirnov test.