Role of RAB27A in Skin Color Determination Figure 4. Melanin content in the epidermis of HSSs is correlated with the amount of RAB27A expression in the original cultured melanocytes. Surface images of HSSs composed of different lines of melanocytes with different expression levels of RAB27A together with same donor-derived keratinocytes and fibroblasts. Melanin contents in the original cultured melanocytes normalized by the amount of protein are shown at the bottom (A). Scale bar = 5 mm. Scatter plots indicate correlations between the expression level of RAB27A protein in the original cultured melanocytes and melanin contents in HSSs (B), and between the number of melanosomes in BKCs and melanin contents in HSSs (C). doi:10.1371/journal.pone.0041160.g004 composed of RAB27A shRNA-lentivirus transfected melanocytes had a lighter skin color (L* = 46.9) compared to those composed of melanocytes treated with the SCR-lentivirus (L* = 42.5) resulting in a significant difference in L* values (DL* = 4.4) (Fig. 6F). The data also showed that the melanin content in the epidermis was significantly lower in HSSs composed of melanocytes treated with the lentiviral vector expressing shRNA for RAB27A (Fig. 6G). Discussion People whose ancestors originated from lower latitude areas tend to have darker skin color, whereas people whose ancestors were from higher latitude areas tend to have relatively lighter skin color, indicating that human skin color is one of the characteristics evolutionarily and geographically acquired to protect the skin from harmful ultraviolet radiation [40,41]. Regarding such correlations, the Fitzpatrick Scale, which represents fair skin people to always or usually sunburn (Types I and II) and dark skin people to be only slightly or never sunburned (Types V and VI), was developed in 1975 to classify skin complexion and its tolerance to sunlight among different skin types and has been widely used for dermatologic research [42]. Apart from the paper demonstrating that the activity of melanosome transfer from melanocytes to keratinocytes is higher in darker skin than in lighter skin [19], we previously demonstrated that the origin of melanocytes predom- inantly determines the skin color rather than the keratinocytes in HSS systems in vivo [43]. Those findings extremely encouraged us to examine which melanocytic factors were predominantly engaged in the determination of skin color focusing on molecules involved in melanin transport processes within melanocytes where mature melanosomes initially bind to kinesin, a microtubule- associated motor protein, and are then directed to the cell periphery, followed by the detachment of melanosomes from microtubles and by their binding to actin filaments through the MYO5A-SLAC2-A-RAB27A complex [4]. In this study, we demonstrated a clear correlation between the expression level of RAB27A and melanin content and the significantly reduced melanosome transfer to keratinocytes and skin color intensity by the siRNA-driven down-regulation of RAB27A. These results consistently illustrated that RAB27A is in charge of the actin- associated melanosome transport within melanocytes and plays an essential role in determining ethnic skin color differences. One important issue addressed in the current study refers to how RAB27A expression is regulated to impact the skin color intensity. It has been reported that the expression of RAB27A is PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e41160