Abstract. Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with ·MSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer. IntroductionHyperpigmentation disorders, such as melasma, postinflammatory hyperpigmentation and lentigo senilis (LS), are associated with abnormal accumulation of melanin pigments, which can be improved by skin lightening reagents (1). Furthermore, Asian women prefer lighter skin color and there is a great demand to develop safer and more effective skin whitening agents (2). Although much effort has been made to develop novel therapeutic agents against pigmentation abnomalities, safer, more effective and less irritating lightening agents are still required.To date, research on the regulation of melanogenesis has focused on factors that can inhibit tyrosinase activity and decrease total melanin content (3). Considerable knowledge of basic pigment distribution and regulation processes has been accumulated to date and should be applicable to the discovery of new targets for developing depigmenting agents.As the present screening methods for depigmenting agents are time-consuming, rapid high-throughput screening (HTS) is required for depigmenting agent screening. Furthermore, HTS of diversified chemical libraries provide a major means for identification of lead candidates for drug or cosmetic ingredient development (4).Visible pigmentation in mammals requires the transfer of melanin granules from melanocytes to keratinocytes. For this intercellular transfer to be effective, melanosomes must first accumulate at the distal end of the melanocyte dendrites using cooperative transport mechanisms (5). These include long range bidirectional, microtubule-dependent melanosome movements along the dendrite length before coupling to myosin Va-dependent capture machinery within the distal actin-rich regions of the dendrite (6). Myosin Va is recruited onto the melanosome surface by a receptor complex containing Rab27a and melanophilin in a Guanosine triphosphate (GTP)-dependent fashion (7-9). The absence of any one of these three proteins collapses the myosin Va-dependent capture of melanosomes in the periphery, causing instead their accumulation in the perikaryon of th...