BLOC-1 function in the formation of endosomal tubules does not require SNARE binding by AP-3 or BLOC-1. (A and B) Ap3d1^−/−/Pldn^−/− melanocytes expressing AP-3δ^WT/Pldn^WT and transiently transfected with mCherry-STX13 and GFP-RAB11 were analyzed by live-cell spinning disk microscopy. (A) Each channel from a frame of a representative image is shown alongside skeletonized tubules; a merged nonskeletonized image is shown at right. Boxed regions are magnified fourfold in the insets. Black or yellow arrowheads, tubules positive for both GFP-Rab11 and mCherry-STX13. Scale bar, 5 µm; inset scale bar, 1 µm. (B) GFP-RAB11–positive tubules per 25-µm² region (10 per cell) were manually counted from skeletonized images of 40 cells and assessed for mCherry-STX13 overlap. mCherry-STX13–positive tubules were similarly quantified and assessed for GFP-RAB11 overlap. Bars represent the percentage (mean ± SEM) of overlapped tubule number relative to total tubule number. (C–G) Pldn^−/− melanocytes or Ap3d1^−/−/Pldn^−/− melanocytes that were untransduced (−) or transduced to express AP-3δ^WT/Pldn^WT, AP-3δ^WT/Pldn^mut1, or AP-3δ^mut1/Pldn^mut1 were transiently transfected with GFP-RAB11 and analyzed by live-cell spinning disk microscopy. (C) A representative frame from an image sequence of the indicated cell type is shown beside the corresponding skeletonized image. Boxed regions are magnified fourfold in the insets. Arrowheads, GFP-RAB11–labeled tubules. Scale bar, 5 µm; inset scale bar, 1 µm. (D) Montages from image streams of Ap3d1^−/−/Pldn^−/− melanocytes expressing AP-3δ^WT/Pldn^WT (top) or AP-3δ^mut1/Pldn^mut1 (bottom) exemplifying tubule lifetimes. Arrowheads, tubules followed through multiple frames. Scale bar, 1 µm. (E–G) GFP-RAB11–labeled tubules in indicated cell samples were quantified as in A for tubule number/25 µm² (E) and length (F) and also for lifetime (the length of time a given tubule could be detected within the image stream; G). Results are displayed as Tukey box-and-whisker plots (10th–90th percentiles); mean is denoted by +. At least five cells were analyzed for each condition. The numbers of tubules quantified for length and lifetime were 278/37 for AP-3δ^WT/Pldn^WT-expressing cells, 326/18 for AP-3δ^WT/Pldn^mut1-expressing cells, 952/31 for AP-3δ^mut1/Pldn^mut1-expressing cells, 322/15 for untransduced Ap3d1^−/−/Pldn^−/− cells, and 379 (tubule length) for Pldn^−/− cells. *, P < 0.05; ***, P < 0.001; n.s, not significant.