Spastizin and AP5Z1 regulate association of lysosomes with motor proteins.

(A) Quantification of the number of tubular lysosomes in wild-type MEFs transfected with a control siRNA or an siRNA down-regulating AP5Z1. Superplot: means and SEM, N = 74 cells from 3 independent experiments. Paired t test on the means. (B) Quantification of the number of tubular lysosomes in wild-type MEFs transfected with a control siRNA and an siRNA down-regulating Spg15. Superplot: means and SEM, N > 92 cells from 4 independent experiments. Paired t test on the means. (C) Proportion of tubular lysosomes moving > 1.2 μm over 1 minute in wild-type MEFs transfected with a control siRNA, an siRNA down-regulating AP5Z1 or Spg15. Superplot: means and SEM, N > 60 cells from 3 different independent experiments. RM one-way ANOVA on the means, Dunnett’s multiple comparisons test. (D) Western blots showing co-immunoprecipitation of spastizin-HA with KIF13A-YFP or mutant KIF13A-ST-YFP (devoid of the motor domain). Input represents 5% of lysate added to the immunoprecipitation assay. (E) Quantification of the number of tubular lysosomes in wild-type MEFs transfected with wild-type KIF13A or mutant KIF13A-ST. Superplot: means and SEM, N > 69 cells from 4 independent experiments. RM one-way ANOVA on the means, Dunnett’s multiple comparisons test. (F) Proportion of tubular lysosomes moving > 1.2 μm over 1 minute in wild-type MEFs transfected with wild-type KIF13A or mutant KIF13A-ST. Superplot: means and SEM, N > 19 cells from 3 different independent experiments. RM one-way ANOVA on the means, Dunnett’s multiple comparisons test. (G) Western blots showing co-immunoprecipitation of p150Glued with GFP-AP5Z1. Input represents 5% of lysate added to the immunoprecipitation assay. (H) Quantification of the number of tubular lysosomes in wild-type MEFs transfected with a vector overexpressing the dominant negative construct p150Glued-CC1-mCherry. Superplot: means and SEM, N > 77 cells from 3 independent experiments. Paired t test on the means. (I) Proportion of tubular lysosomes moving > 1.2 μm over 1 minute in wild-type MEFs transfected with the dominant negative construct p150Glued-CC1-mCherry. Superplot: means and SEM, N > 65 cells from 3 different independent experiments. Paired t test on the means. The raw data underlying panels A, B, C, E, F, H, and I can be found in S1 Data file.