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. 2023 Nov 22;44(13):e0374232023. doi: 10.1523/JNEUROSCI.0374-23.2023

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Copyright © 2024 Lombino et al.

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Figure 7. — Functional inhibition of katanin reduces microtubule polymerization into dendritic spines. A, Immunostaining of endogenous EB3 (blue) following expression of td-Tomato (volume marker, red) together with either GFP or GFP-p60DEID (green channel not shown), DIV14 neurons. B, Quantification of % of EB3-positive spines with heads. GFP: 8.81 ± 1.35%, GFP-p60DEID: 4.90 ± 1.53%, Mann–Whitney U test p = 0.0345 (GFP: n = 33 cells, 979 spines; GFP-p60DEID: n = 20 cells, 402 spines). C, Quantification of percentage of EB3-positive stubby spines. GFP: 8.18 ± 1.50%, GFP-p60DEID: 4.86 ± 1.87%, Mann–Whitney U test, p = 0.1059 (GFP: n = 33 cells, 505 spines; GFP-p60DEID: n = 20 cells, 187 spines). D, Quantification of percentage of EB3-positive filopodia. GFP: 9.52 ± 2.90%, GFP-p60DEID: 15.26 ± 7.43%, Mann–Whitney U test, p = 0.9332 (GFP: n = 33 cells, 137 spines; GFP-p60DEID: n = 20 cells, 59 spines), three independent experiments. E, Time-lapse microscopy to investigate EB3 invasion into spines (compare with movie 7-1). Please note that the signal intensity of this example is shown with increased brightness to visualize the transient invasion and removal of the red EB3 signal (boxed region). F–H, Quantification of time-lapse video microscopy using EB3-Tomato. Please note the imaging period was 10 min, four independent experiments. F, Percentage of spines with heads invaded by EB3-Tomato over 10 min: GFP: 15.16 ± 4.43%, GFP-p60DEID: 4.54 ± 2.01%, Mann–Whitney U test, p = 0.0487 (GFP: n = 12; GFP-p60DEID: n = 13 cells). G, Percentage of stubby spines invaded by EB3-Tomato over 10 min: GFP: 12.90 ± 5.29%, GFP-p60DEID: 11.93 ± 4.51%, Mann–Whitney U test, p = 0.9794 (GFP: n = 12; GFP-p60DEID: n = 13 cells). H, Percentage of filopodia invaded by EB3-Tomato over 10 min: GFP: 22.07 ± 3.58%, GFP-p60DEID: 27.78 ± 5.82%, Unpaired two-tailed t test p = 0.4211 (GFP: n = 12; GFP-p60DEID: n = 13 cells), four independent experiments.