Fig. 2: The Tuba4a mutation reduces overall tubulin polyglutamylation levels but is still incorporated into microtubules. | Nature Communications

Fig. 2: The Tuba4a mutation reduces overall tubulin polyglutamylation levels but is still incorporated into microtubules.

From: Disruption of tubulin-alpha4a polyglutamylation prevents aggregation of hyper-phosphorylated tau and microglia activation in mice

Fig. 2

A Relative Tuba4a mRNA expression levels. (+/+) set to 1 (gray dotted line) in adult mice. Median (center), interquartile range (bounds of boxes), and minima and maxima (whiskers) are indicated. n = 11 samples per genotype. Two-sided alternative hypothesis (H1) testing was used to assess statistical significance. B Representative western blot analysis depicting tubulin polyglutamylation (polyGlu), Tuba4a, total alpha-tubulin, NSE and Actin in adult Tuba4aΔpolyGlu (+/+), (+/p) and (p/p) mice. Please note that the remaining levels of polyglutamylation are due to tubulins other than Tuba4a. C, D Quantification of Tuba4a normalized to NSE (tubulin-independent loading control) (C) and polyglutamylated tubulin normalized to total alpha-tubulin (D) signal intensities shown in (B). (+/+) set to 100%. C n = 4(+/+ and p/p), 3(+/p) and D n = 5(+/+ and p/p), 4(+/p) experiments per genotype. E Developmental expression of polyGlu, Tuba4a, Tubb3, and Actin in hippocampal neurons at different DIV, as indicated. F, G Quantification of Tuba4a (F) and polyglutamylated tubulin (G) signal intensities shown in (E) over time. Two-way ANOVA (DIV × genotype) with p = 0.2 (Tuba4a) and p = 0.0004 (polyGlu), n = 3 independent cultures per genotype per time point. H Immunohistochemical analysis of Tuba4a (green) and polyGlu (greyscale or blue) in CA1 region of hippocampal brain sections derived from 12-month-old Tuba4aΔpolyGlu (+/+) and (p/p) mice. Scale bar, 50 µm. I Quantification of polyGlu normalized to the area analyzed, shown in (H). +/+ set to 1; n = 3 mice per genotype. J Representative coIP of Tuba4a and Tubb3 from hippocampal lysates derived from adult mice. In addition, polyglutamylated tubulin levels were analyzed. K Quantification of signal intensities of precipitated polyGlu normalized to signal intensities of precipitated Tuba4a, shown in (J), n = 3 experiments. L Coimmunostaining of Tuba4a (green) and AnkyrinG (blue; axonal marker) using DIV14 neurons. n = 3 experiments. Scale bars, 20 µm (overview), 5 µm (soma), 2.5 µm (axon). M High-resolution immunogold electron microscopy (EM) of Tuba4a in the medulla oblongata (rich in parallel axons) from adult Tuba4aΔpolyGlu (+/+) and (p/p) mice. Micrographs on the left show magnifications of the white rectangles. The presence of myelin sheets (#) identifies axons. Note, the pearl necklace-like distribution of gold particles at microtubules. Scale bars, 500 nm (overview), 200 nm (magnification). n = 3 mice per genotype. Two-sided unpaired Student’s t-test (A, C, D, I, K) and ANOVA (F, G) were used to assess statistical significance. *p < 0.05, ***p < 0.001. Data represent mean ± SEM, if not stated otherwise. Source data, including exact p-values, are provided as a Source Data file.

Back to article page