Fig. 1
Phosphorylation of Thr211 residue of SEPT3 delocalizes it from the dendritic spine base and promotes sER extension into spines in cooperation with activated MYO5A
a, Co-IP with IgG or anti-SEPT3 antibodies using protein extracted from the hippocampal DG of Sept3+/+ and Sept3−/− male mice, followed by IB analysis using antibodies against SEPT3, α-Tubulin, and phosphoserine/threonine (pSer/Thr). Arrowhead indicates the SEPT3 signal.
b, Left and middle, Co-IP with anti-SEPT3 antibody using protein extracted from the hippocampal DG of Sept3+/+ male mice at 0 min and 10 min after ECS. Right, Normalized phosphorylation level of SEPT3 on Ser/Thr. n = 3 experiments; two-tailed unpaired t test.
c, Top, Representative images of dendritic spines and sER in primary cultured rat DG granule cells expressing wild-type (WT) or phosphomimetic mutants of SEPT3, showing the morphology of spines (cyan) and sER (magenta) at 21 days in vitro (DIV). Scale bar, 5 μm. Middle, Spine volume, defined as the total fluorescence intensity of the spine within regions of interest (ROIs) divided by the average fluorescence intensity of the dendrite within ROIs. Bottom, Percentage of spines with sER. n = 15 dendrites; one-way ANOVA with Tukey’s multiple comparisons test.
d, Left, Representative images of dendritic spines and GFP-tagged SETP3-WT, non-phosphorylatable alanine substitution mutant (T211A), or phosphomimetic aspartic acid mutant (T211E). White squares indicate the regions used for analysis. In the magnified images, white dashed lines outline the boundary between the cytosol and sER. Scale bar, 0.5 μm. Right top, Quantification of sER area as a percentage of the total region of interest (ROI). Right bottom, Quantification of the proportion of GFP-SEPT3 fluorescence intensity (IF) within the sER area relative to the total ROI. n = 30 spines; one-way ANOVA with Tukey’s multiple comparisons test.
e, Top, Representative images of dendritic spines and sER from DG granule cells overexpressing SEPT3-WT or T211E, transiently co-expressing either MYO5A-WT or CCtr (the functionally active form). Scale bar, 5 μm. Middle, Spine volume. Bottom, Percentage of spines with sER. n = 15 dendrites; one-way ANOVA with Tukey’s multiple comparisons test.
f, Top, Representative images of dendritic spines and sER from DG granule cells expressing shSEPT3, an shRNA resistant mutant of SETP3-WT, T211E, or T211A, and/or co-expressing MYO5A-WT or CCtr. Scale bar, 5 μm. Middle, Spine volume. Bottom, Percentage of spines with sER. n = 14–17 dendrites; one-way ANOVA with Tukey’s multiple comparisons test.
Data are mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significantly different.