The GDP-bound state of SEPT9 recruits dynein and induces retrograde transport. (A) Coomassie-stained gel (top) and Western blot (bottom; anti-His) show binding of the G domain of SEPT9 to GST-DIC(108–268) in the presence of GDP, GTP, or GTPγS (0.5 mM). Quantification shows the relative ratios of SEPT9 G domain to GST-DIC(108–268) protein band intensities from three independent experiments. (B) Gels show Western blots (anti-GST/anti-His) of pull-downs of recombinant His-tagged wild-type (wt) or GTPase-dead (T339G) SEPT9 with GST or GST-DIC(108–268). Bar graph shows the relative ratio of the wild-type or GTPase-dead (T339G) G domain of SEPT9 to GST-DIC(108–268) from four independent experiments. Error bars indicate SEM. (C) Images show DAPI-stained COS-7 cells transfected with mitochondria-targeted (Mito-TagRFP) wild-type and GTPase-dead (T339G) SEPT9. Scale bars, 20 µm. (D) Bar graph shows perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total mitochondria per cell (n = 24–27). Error bars indicate SEM. (E) Images show COS-7 cells transfected with mitochondria-targeted (MitoTagRFP) wild-type or GTPase-dead (T339G) SEPT9 and stained with an antibody against DIC. Scale bars, 20 µm. (F) Plot shows DIC fluorescence intensity (mean ± SEM; Mann–Whitney U test) on mitochondria with SEPT9-Mito-TagRFP or SEPT9(T339G)-Mito-TagRFP per cell (n = 40–41). (G) Images show the distribution of lysosomes (LAMP-1) in COS-7 cells transfected with wild-type SEPT9-mCherry or SEPT9(T339G)-mCherry (insets). Scale bars, 20 µm. (H) Plot shows the ratio (mean ± SEM; unpaired t test) of perinuclear to peripheral LAMP1 fluorescence intensity per cell (n = 21–22). **, P < 0.01; ***, P < 0.001. A.U., arbitrary units.