Figure 4.

Binding of either AP-3δ to VAMP7 or Pldn to STX13 is required for BLOC-1–dependent VAMP7 localization to melanosomes. (A–E) Ap3d1^−/−/Pldn^−/− melanocytes that were untransduced (-; A) or stably transduced with the indicated WT (black text) or SNARE binding mutant (magenta text) AP-3δ and Pldn variants (B–E) were transiently transfected with GFP-VAMP7, fixed, immunolabeled for STX13, and analyzed by dIFM and brightfield (BF) microscopy. Shown are corresponding BF images (left), individual labels for GFP-VAMP7 and STX13, merged images of GFP-VAMP7 and STX13, and overlap of GFP-VAMP7 with pigment granules (if any) pseudocolored magenta from the BF image (right). Boxed regions are magnified sevenfold in the insets. White arrows, overlap of GFP-VAMP7 with STX13; cyan arrows, overlap of GFP-VAMP7 with pigment granules; white arrowheads, lack of overlap of GFP-VAMP7 with STX13. The cell outline is shown in the BF images in A and E. Scale bar, 10 µm; inset scale bar, 1 µm. (F) Quantification (mean ± SEM) of percentage GFP-VAMP7 overlap with STX13 across 18 cells for each sample over three separate experiments. ****, P < 0.0001; n.s., not significant.