Figure 2.

Altered microtubule structure and dynamics in sacsin KO cells

(A) Confocal immunofluorescent images of sacsin WT/KO cells stained for vimentin, and the MTOC marker γ-tubulin. Arrowheads point to the most intense signal in each cell, showing that vimentin bundles surround the MTOC in sacsin KO cells. Scale bar, 10 μm.

(B) Confocal images of immunostaining for α-tubulin, neurofilament heavy, and acetylated tubulin in WT and sacsin KO cells. Arrowheads mark coincidence of acetylated tubulin and neurofilament bundles, suggesting that acetylated tubulin structures are found in proximity to neurofilament bundles, but also localize throughout the cell. Scale bar, 10 μm.

(C and D) Quantification of images in (B). n = 3, SEM, Student’s t test.

(E) Confocal images of WT/KO cells treated with nocodazole (NDZ) labeled for α-tubulin and acetylated tubulin at indicated time points following nocodazole washout. Scale bar, 10 μm.

(F) Quantification of (E). n = 3 coverslips, SEM, one-way ANOVA with Tukey’s post test.

(G) Representative TIRF microscopy images from WT and sacsin KO cells expressing EB1-GFP. Microtubule growth tracks are color coded marking their position over time. Insets show the enlargement of outlined regions and movement of individual comets over time (circles). Numbers refer to seconds.

(H) Quantification of microtubule polymerization velocity marked by EB1-GFP movement in WT/KO cells (F and Video S1). n = 34 WT and n = 25 sacsin KO cells from three independent experiments, Student’s t test, ^∗∗p < 0.01.