Figure 8. BLOC-2 facilitates contacts of recycling endosomal tubules with maturing melanosomes. WT melan-Ink4a and BLOC-2− cells stably expressing EGFP-STX13 were transfected with mRFP-OCA2 and imaged in a single plane at 1 fps for 5 min by spinning disk microscopy. (a) Starting frame from representative image series (Videos 3 and 5). Boxed region is magnified 2× in inset. Bar, 10 µm. (b) Frames from the image series of the magnified boxed region in a at the times (seconds) indicated in top left. Arrows, melanosomes that interact with tubules; arrowheads, examples of tubules. (c) EGFP-STX13 labeling in a single panel highlighted in b was analyzed using the ImageJ “skeletonize” function to emphasize tubule length (Videos 4 and 6 for image series). (d) The number of EGFP-STX13 tubules (branched structures in skeletonized images) per video frame using a constant frame size (12 × 12 µm) was quantified within at least five WT and BLOC-2− cells in each of three independent experiments. Shown is the mean number of tubules (±SD) identified in each frame. (e) The length of EGFP-STX13 tubules between vertices in skeletonized images was quantified within 10 WT and BLOC-2− cells each in three independent experiments. Shown are the percentages (means ± SD) of tubules with lengths shorter or longer than 1 µm. (f) The percentage of EGFP-STX13 tubules that contact mRFP-OCA2–positive compartments was quantified using a MATLAB program and averaged from five WT and BLOC-2− cells each in six independent experiments. Shown are the mean values ± SD. (g) For those EGFP-STX13 tubules that contact mRFP-OCA2 compartments, the duration of contact was quantified. Shown are the percentages of contacts (means ± SD) that lasted for greater or less than 20 s. *, P < 0.05; **, P < 0.01.
Figure 8.

BLOC-2 facilitates contacts of recycling endosomal tubules with maturing melanosomes. WT melan-Ink4a and BLOC-2− cells stably expressing EGFP-STX13 were transfected with mRFP-OCA2 and imaged in a single plane at 1 fps for 5 min by spinning disk microscopy. (a) Starting frame from representative image series (Videos 3 and 5). Boxed region is magnified 2× in inset. Bar, 10 µm. (b) Frames from the image series of the magnified boxed region in a at the times (seconds) indicated in top left. Arrows, melanosomes that interact with tubules; arrowheads, examples of tubules. (c) EGFP-STX13 labeling in a single panel highlighted in b was analyzed using the ImageJ “skeletonize” function to emphasize tubule length (Videos 4 and 6 for image series). (d) The number of EGFP-STX13 tubules (branched structures in skeletonized images) per video frame using a constant frame size (12 × 12 µm) was quantified within at least five WT and BLOC-2− cells in each of three independent experiments. Shown is the mean number of tubules (±SD) identified in each frame. (e) The length of EGFP-STX13 tubules between vertices in skeletonized images was quantified within 10 WT and BLOC-2− cells each in three independent experiments. Shown are the percentages (means ± SD) of tubules with lengths shorter or longer than 1 µm. (f) The percentage of EGFP-STX13 tubules that contact mRFP-OCA2–positive compartments was quantified using a MATLAB program and averaged from five WT and BLOC-2− cells each in six independent experiments. Shown are the mean values ± SD. (g) For those EGFP-STX13 tubules that contact mRFP-OCA2 compartments, the duration of contact was quantified. Shown are the percentages of contacts (means ± SD) that lasted for greater or less than 20 s. *, P < 0.05; **, P < 0.01.

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