Binding of either AP-3δ to VAMP7 or Pldn to STX13 is required for STX13 sorting into BLOC-1–dependent tubules. Ap3d1^−/−/Pldn^−/− melanocytes that were either untransduced (-) or stably transduced to express the indicated WT (black) or SNARE binding mutant (magenta) variants of AP-3δ and Pldn were transiently transfected with mCherry-STX13 and analyzed by live-cell spinning disk microscopy for tubule formation. (A) Top panels: Individual frames from representative image streams. Bottom panels: Binary skeletonized images of the top panels. Boxed regions are magnified 6.5-fold in the insets. Arrowheads, STX13-positive tubules. Scale bar, 5 µm; inset scale bar, 1 µm. (B and C) STX13-positive tubules in ten 25-µm² regions from skeletonized images of each of at least five analyzed cells per condition in each of three separate experiments were analyzed. (B) Tubule number per 25-µm² region was quantified. (C) Length was quantified for 1,268 tubules in AP-3δ^WT/Pldn^WT-expressing cells, 1,247 tubules in AP-3δ^WT/Pldn^mut-expressing cells, 1,190 tubules in AP-3δ^mut1/Pldn^mut1-expressing cells, and 303 tubules in untransduced cells. Data in B and C are represented by Tukey box-and-whisker plots (10th–90th percentiles); mean is denoted as +. ***, P < 0.001.