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

Figure 6.

Figure 6

Characterization of protein localization, dendritic spine density and morphology in DIV14-15 hippocampal neurons expressing EGFP and mCherry (control), myosin-IIb-mCherry or mutated myosin-IIb-Y265C-mCherry. (A) Representative maximum projections of confocal Z-stacks for EGFP, mCherry, and wild-type and mutated myosin-IIb localization. Scale bars 4 μm. (B) The quantification of protein localization revealed similar enrichment for both wt and mutated myosin-IIb to dendritic spines. Spine-to-dendrite fluorescence intensity ratios: control: 0.93 (n = 17 cells, 170 spines), myosin-IIb: 1.73 (n = 17 cells, 170 spines), myosin-IIb-Y265C 1.79 (n = 18 cells, 180 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, myosin-IIb-mCherry, or myosin-IIb-Y265C-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 = 0.2, stubby = 0.1, total = 0.55 spines/μm (n = 17 neurons, 1,825 spines, 3,354 μm dendrite); myosin-IIb: thin = 0.27, mushroom = 0.2, stubby = 0.09, total = 0.57 spines/μm (n = 17 neurons, 1,718 spines, 3,459 μm dendrite); myosin-IIb-Y265C: thin = 0.27, mushroom = 0.22, stubby = 0.12, total = 0.61 spines/μm (n = 18 neurons, 2,253 spines, 3,877 μm dendrite). Data is pooled from 2 experiments and represented as mean ±SEM. Significant differences were not detected by one-way ANOVA test with Games-Howell or 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), myosin-IIb-mCherry or myosin-IIb-Y265C-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.