VAMP7 is a BLOC-1 cargo. (a–j) BLOC-1–deficient melan-pa (BLOC-1−/−; a–e) or “rescued” melan-pa cells stably expressing myc-Pallidin (BLOC-1R; f–j) and transiently expressing GFP-VAMP7 (green) and mCh-STX13 (red) were fixed and labeled for TYRP1 (cyan) and analyzed by deconvolution immuno-FM. Arrowheads show GFP-VAMP7 and TYRP1 retained in mCh-STX13–positive endosomes, and arrows show melanosomes with GFP-VAMP7 and TYRP1 but lacking mCh-STX13. Insets of boxed regions are magnified five times. (k–m) WT melan-Ink4a (n–p) and BLOC-1–deficient melan-pa (BLOC-1−/−) transiently expressing GFP-VAMP2 (green) and mCh-STX13 (red, k and n), VAMP4-HA (green, l and o), or GFP-VAMP8 (green, m and p) were fixed, labeled for HA (green) and Giantin (red, l and o) or for LAMP1 (red, m and p), and analyzed by deconvolution immuno-FM. Merged images are shown with BF images pseudocolored blue. Dashed line in o indicates cell borders. Insets are boxed regions magnified six times. (q–v) BLOC-1−/− melan-pa melanocytes transiently transfected with GFP-VAMP7 (green), mCh-STX13 (red), and either myc-Pallidin (“rescue”) or myc-Muted (“mock rescue”) for indicated times. Arrowheads show GFP-VAMP7 retained in mCh-STX13 endosomes; and arrows point to GFP-VAMP7 in melanosome precursors. Note that pigmentation is not detected until ∼72 h after transfection with myc-Pallidin. Insets are boxed regions magnified 3.5 times. (u) Quantification (mean ± SD from 15 regions of at least nine cells per time point representing three independent experiments) of GFP-VAMP7–labeled structures that lack mCh-STX13 in rescue and mock rescue cells at 12, 24, and 48 h after transfection. (v) Area (mean ± SD from at least 15 cells per time point representing four independent experiments) of overlap between GFP-VAMP7 and mCh-STX13 in the periphery of mock rescue and rescue cells quantified at 12, 24, and 48 h after transfection. **, P < 0.01; ****, P < 0.0001. BF images in d, I, and k–t are pseudocolored blue in merge. Bars: (a–t) 10 µm; (insets) 2 µm.
