Spastin promotes endosomal tubule fission at ER tubules. (A) Stills from Video 1 of MRC5 cells stably expressing GFP-SNX1, showing tubule fission in control cell or cell depleted of spastin by siRNA knockdown (KD). The main panels show an overview of the cell; the small panels illustrate a single fission event. Yellow arrowheads track the tip of the elongating endosomal tubule and the fissioned domain. Time 0 corresponds to the frame in which the tubules first emerge. Corresponding histograms show mean tubule duration (left), mean percentage of tubules with different fates (middle), and duration of tubules with each fate (right). left, n = 6 experiments; middle and right, n = 5; see Materials and methods for numbers of tubules analyzed in these and subsequent live cell experiments. Bars in small panels, 2 µm. (B) Stills from Video 3 of MRC5 cells stably expressing GFP-SNX1 and transiently expressing RFP-KDEL, showing mock-transfected (top) or spastin KD (bottom) cell. The main panels show an overview of the cell; the small panels illustrate a single fission event. White arrowhead marks approximate site of fission. Time 0 corresponds to the frame in which the tubules first emerge. Bars in small panels, 1 μm. The corresponding histograms show percentage of fission events occurring at site of ER tubule overlap (C) and the duration of overlap between ER and endosomal tubule before breakage or collapse (D); n = 3 experiments. (E) EM of early endosome (EE) and multivesicular body (MVB) structures in MEFs from spastin^wt/wt (WT), spastin^wt/N384K (HET), and spastin^N384K/N384K (KI) animals. Arrows indicate points of ER contact, and the mean number of contacts is quantified (pooled, EE and MVB results combined). n = 3 experimental repeats. See Materials and methods for number of structures analyzed. Bars: (light micrographs, large panels) 10 µm; (EM) 500 nm. All histograms show mean ± SEM. P-values generated by two-tailed Student’s t test (A, C, and D) or ANOVA for effect of genotype (E).