Spastin–IST1 interaction drives endosomal tubule fission. (A) Wild-type HeLa cells or HeLa cells stably expressing Myc-M1-spastin were imaged by confocal immunofluorescence microscopy (IF) with the antibodies indicated. Arrowheads indicate colocalized puncta. In cells labeled with endogenous spastin, soluble cytosolic signal was removed using a prefixation cytosol extraction buffer. (B) MRC5 cells stably expressing GFP-M1 spastin and mCherry-SNX1 were labeled with IST1 and GFP antibodies and imaged with Airyscan IF. Small panels show individual channels. Arrowheads indicate a region of colocalization between IST1 and spastin at the base of a SNX1 tubule. (C) Wild-type HeLa cells or cells expressing the siRNA-resistant proteins indicated (see schematic) were subjected to endogenous IST1 KD, then fixed and visualized by IF for endogenous SNX1. Mean ± SEM number per cell of SNX1 tubules >2 µm long is shown in the histogram (n = 5). Bars: (A and C, main image) 10 µm; (A and C, magnified insets) 5 µm; (B) 500 nm. P-values generated by two-tailed Student’s t test.