ATPase activity and interaction with ESCRT-III are required for spastin to regulate endosomal tubulation. (a–d) Wild-type HeLa cells (a), HeLa cell lines stably expressing myc-tagged M87 spastin (b), myc-tagged M87 spastinK388R (c), or myc-tagged M87 spastinF124D (d) were subjected to mock transfection or were transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant) or with siRNA oligonucleotides directed against endogenous and exogenous spastin (spastin 1 and 6). Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting (Fig. S3). The number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in e; n = 3 independent experiments. (f–h) GFP-tagged VPS4-E235Q was transiently transfected into HeLa cells stably expressing myc-tagged wild-type (wt) M87 spastin (f) or myc-tagged M87 spastinF124D (g), which has dramatically reduced binding to CHMP1B and IST1. The cells were labeled with anti-myc antibodies. The extent of colocalization between GFP-VPS4-E235Q and the spastin proteins was estimated by calculating the Pearson’s correlation coefficient for red and green pixels in each cell, using Volocity software (h; n = 3 experiments, 20 cells per condition quantified in each experiment). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.