Figure 1.

Disease-associated spastin MIT mutants cannot support normal endosomal tubule fission. (A) Schematic diagram of spastin's domain structure, annotated with the position of artificial (above) and disease-associated (below) mutants used in this study. HR, hydrophobic region; MIT, MIT domain; MTB, microtubule binding domain; AAA, ATPase domain. (B–F) Wild-type HeLa cells or cells expressing the siRNA-resistant spastin constructs indicated were subjected to mock transfection or siRNA knockdown (KD) of endogenous spastin, then fixed and labeled for endogenous SNX1. Insets show higher magnification views of the boxed areas. Scale bar = 10 μm. (G) The percentage of cells with the longest SNX1 tubule >2 μm was counted (n = 100 cells per condition). Mean and SEM are shown for three biological repeats. P-values were calculated using one-way ANOVA with Bonferroni's post hoc multiple comparison test. In addition to the p-values shown, the M87-wild-type KD value was significantly different from all other KD values, with a significance of at least p < 0.05. (H) Depletion of spastin and expression of siRNA resistant constructs was confirmed by Western blotting with the antibodies indicated. GAPDH labeling is shown to verify equal sample loading.