Role of RAB27A in Skin Color Determination Figure 1. The number of melanosomes transferred to BKCs in the skin obtained from African-descent and from Caucasian individuals. Ultrastructural images of BKCs from African-descent subject-derived breast skin (A) and upper inner arm skin (B) and from Caucasian subject-derived abdominal skin (C) and upper inner arm skin (D). Fifty-one, thirty-one, forty-eight and thirty-one BKCs were imaged to calculate the numbers of melanosomes in those cells, respectively. N = nucleus, broken lines = basal membranes, and arrowheads point to melanosomes. Scale bars = 5 mm. The numbers of melanosomes transferred to BKCs is shown in (E). The values reported represent means 6 SD. Statistical significance was evaluated by the Kruskal-Wallis test; asterisks indicate statistical significance (p,0.01). doi:10.1371/journal.pone.0041160.g001 More melanosomes from dark skin-derived melanocytes with higher RAB27A expression are transferred to keratinocytes than from light skin-derived melanocytes, resulting in higher melanin content in HSSs To further examine the contribution of RAB27A expression to the amount of melanosome transfer and consequently to the intensity of skin pigmentation, melanocytes derived from three dark skin subjects and from four light skin subjects were co- cultured with keratinocytes from a light skin subject. The difference in RAB27A expression level between the two groups is shown in Fig. 3A. After the co-culture of keratinocytes with dark skin-derived melanocytes for 5 days, transferred melanosomes were easily detected with a specific antibody against the melanosome protein gp100 in adjacent areas of keratinocyte nuclei (Fig. 3B), in contrast to rarely observed melanosomes in keratinocytes co-cultured with light skin-derived melanocytes (Fig. 3C). The ratio of melanosome-incorporated keratinocytes to total keratinocytes was significantly higher when the keratino- cytes were co-cultured with dark skin-derived melanocytes expressing higher levels of RAB27A (Fig. 3D). Following co- culture-based assessment, HSSs in vivo were generated by separately mixing 10 cell lines of melanocytes correlating their melanin contents with RAB27A expression level together with identical cell lines of light-skin derived keratinocytes and fibroblasts. The intensity of HSS surface color was found to be dominantly regulated by the ability to synthesize melanin in the original melanocyte populations before they were used in the HSS (Fig. 4A). Consistently, the melanin content in HSS epidermis correlated with the expression level of RAB27A in the melanocytes prior to the HSS generation (Fig. 4B) and with the number of melanosomes transferred to BKCs (Fig. 4C). Effect of suppressing RAB27A expression on melanosome transfer and on skin color For the direct evaluation of the contribution of RAB27A expression to melanosome transfer to keratinocytes and to the resulting skin color, melanocytes treated with a lentivirus-driven RAB27A shRNA were used for melanocyte-keratinocyte co- cultures and the subsequent development of HSSs. Knockdown of RAB27A expression in melanocytes was confirmed by Western blotting analysis in cells treated with the RAB27A shRNA- lentivirus compared to cells treated with the scrambled RNA (SCR)-lentivirus after blasticidin selection for 3 wks (Fig. 5A). Even though there was no noticeable difference in cell shape between cells treated with either type of lentivirus (Fig. 5B, 5C), melanosome aggregation around the nuclei was observed only in melanocytes treated with the RAB27A shRNA-lentivirus (Fig. 5C) when compared to control cells treated with the SCR-lentivirus (Fig. 5B). Immunohistochemistry using an antibody specific for the melanosomal protein gp100 demonstrated that melanosome transfer to surrounding keratinocytes was remarkably reduced in co-cultures with RAB27A shRNA-lentivirus treated melanocytes (Fig. 5E). In contrast, substantial melanosome transfer was observed in keratinocytes co-cultured with SCR-lentivirus treated melanocytes (Fig. 5D). The ratio of melanosome-containing keratinocytes to total keratinocytes was significantly lower in PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41160