Abstract
Melanin incorporated into keratinocytes plays an important role in photoprotection; however, abnormal melanin accumulation causes hyperpigmentary disorders. To understand the mechanism behind the accumulation of excess melanin in the skin, it is essential to clarify the spatial distribution of melanosomes or melanin in the epidermis. Although several markers have been used to detect melanosomes or melanin, no suitable markers to determine the precise localization of melanin in the epidermis have been reported. In this study, we showed that melanocore-interacting Kif1c-tail (M-INK), a recently developed fluorescent probe for visualizing mature melanosomes, binds to purified melanin in vitro, and applied it for detecting melanin in human skin tissues. Frozen skin sections from different phototypes were co-stained for the hemagglutinin (HA)-tagged M-INK probe and markers of melanocytes or keratinocytes, and a wide distribution of melanin was observed in the epidermis. Analysis of the different skin phototypes indicated that the fluorescent signals of HA-M-INK correlated well with skin color. The reconstruction of three-dimensional images of epidermal sheets enabled us to observe the spatial distribution of melanin in the epidermis. Thus, the HA-M-INK probe is an ideal tool to individually visualize melanin (or melanosome) distribution in melanocytes and in keratinocytes in skin tissues.
Highlights
Melanin is one of the main factors responsible for human skin color
We reported that melanin-containing melanosomes in cultured melanocytes were detected with purified T7-glutathione S-transferase (GST)-tagged melanocore-interacting Kif1c-tail (M-INK) [13]
These cells were incubated with HA-M-INK-containing cell lysates, and HA-M-INK signals were visualized with anti-HA tag antibody
Summary
Melanin is one of the main factors responsible for human skin color. It is synthesized by melanogenic enzymes such as tyrosinase within melanosomes, which are specialized organelles in melanocytes, and is transported to and distributed in neighboring keratinocytes. One example of this is the use of Fontana–Masson silver staining, which produces black silver spots that are reaction products of the reducing group of melanin [5] This approach has been widely used for detecting melanosomes or melanin histochemically [6,7,8]. It is impossible to use this approach to observe stained spots together with several proteins simultaneously, and to analyze their three-dimensional (3D) distribution by confocal microscopy Another method widely used for detecting melanosomes or melanin histochemically is immunostaining for Pmel ( known as Pmel and gp100), a transmembrane glycoprotein present in melanosomes [10]. Pmel and melanogenic enzymes are inappropriate markers to monitor the series of processes of melanosome transfer, dispersion, and degradation in keratinocytes
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