Fig. 7: Commander is important for the trafficking of melanogenic enzymes, PMEL processing, compartment neutralisation and dynamic endosomal actin regulation. | Nature Communications

Fig. 7: Commander is important for the trafficking of melanogenic enzymes, PMEL processing, compartment neutralisation and dynamic endosomal actin regulation.

From: Identification of a RAB32-LRMDA-Commander membrane trafficking complex reveals the molecular mechanism of human oculocutaneous albinism type 7

Fig. 7

A Parental MNT1 cells or RAB32 KO, LRMDA KO, SNX17 KO, VPS35L KO, CCDC22 KO or FAM21 KO cells were immuno-stained for TYRP1 and LAMP1. Scale bar corresponds to 20 µm. n = 3 (parental – 67 cells; RAB32 KO – 56 cells; LRMDA KO – 56 cells; SNX17 KO – 56 cells; VPS35L KO – 57 cells; CCDC22 KO – 55 cells, FAM21 KO – 55 cells), large circles represent averages of each independent experiment, and small circles the data for individual cells, colouring corresponds to different independent replicates. 1-way ANOVA with Dunnett’s multiple comparison test on data for replicate averages, data presented as mean values and error bars represent s.d. B Total PMEL levels and levels of processed PMEL fragments were analyzed in whole-cell lysates from parental MNT1 cells and RAB32 KO, LRMDA KO, SNX17 KO, VPS35L KO, CCDC22 KO or FAM21 KO cells. n = 3 biological replicates, 2-way ANOVA with Dunnett’s multiple comparison test, data presented as mean values and error bars represent s.d.; n.s. denotes changes with p > 0.05. C Parental MNT1 cells or RAB32 KO, LRMDA KO, SNX17 KO, VPS35L KO, CCDC22 KO or FAM21 KO cells were stained with LysoTracker. Fixed cells were observed under a confocal microscope. Representative images are shown; the experiment was performed in three independent replicates. Scale bar corresponds to 20 µm. D Parental MNT1 cells or RAB32 KO, LRMDA KO, SNX17 KO, VPS35L KO, CCDC22 KO or FAM21 KO MNT1 cells were treated with DMSO or 100 nM Bafilomycin A1 for 24 h to prevent over-acidification. E Cells from (D) were used for melanin content measurements. n = 4 biological replicates, 2-way ANOVA with Šídák’s multiple comparisons test, data presented as mean values and error bars represent s.d.; n.s. denotes changes with p > 0.05. F Parental MNT1 cells or RAB32 KO, LRMDA KO, SNX17 KO, VPS35L KO, CCDC22 KO or FAM21 KO cells were fixed and immuno-stained for EEA1, FAM21 and F-actin using Phalloidin dye. Representative images are shown; the experiment was performed in three independent replicates. Scale bar corresponds to 20 µm.

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