BLOC-3 is required to retrieve VAMP7 from melanosomes. (a–d) WT melan-Ink4a (Ink4a; a), melan-le (le; b), melan-le:HPS1 (le:HPS1; not depicted), or melan-le:HPS4 (le:HPS4; c) cells transiently transfected with GFP-VAMP7 were analyzed by spinning-disk confocal microscopy 24 h later. Images were acquired at ∼1 fps and a segment of the image sequence is shown. Elapsed time (in seconds) is indicated at lower right. Bar, 2 µm. Arrows show GFP-VAMP7–labeled structures exiting GFP-VAMP7–labeled melanosomes. (d) Fission of GFP-VAMP7 transport intermediates from melanosomes was quantified by counting events within 26.5-µm2 regions in the cell periphery during 5-min image sequences acquired at 1 fps. At least 15 regions from a minimum of six cells, representing three independent experiments, were quantified for each cell type; shown are mean values ± SD. ****, P < 0.0001. (e) Model for VAMP7 cycling during melanosome biogenesis.
