The effective transfer of melanosomes, specialized organelles for melanin production, from melanocytes to keratinocytes, is an absolute requirement for photoprotection in the skin. Melanocytes are pigment producing cells that reside in the basal layer of the epidermis, and form multiple long dendritic processes that transport melanosomes from the melanocyte cell body to the dendritic tips, and then to keratinocytes. Dendrite formation and melanosome transfer to keratinocytes is stimulated by hormones including alpha-melanocyte stimulating hormone, endothelin-1 and nerve growth factor, as well as ultraviolet light, but the second messenger pathways involved in dendrite formation and in melanosome transfer are poorly understood in mammalian models. The formation of dendritic processes in melanocytes is closely linked to reorganization of the actin cytoskeleton, which results in extension of dendrites through the formation of lamellipodia, and transport of melanoses to the dendrite tip. The long term goal of our research efforts is to determine 1) the upstream signaling intermediates involved in dendrite formation and 2) the molecular mediators and regulatory factors involved in melanosome transfer. Rac1 is a monomeric GTP- binding protein that orchestrates actin reorganization with the formation of lamellipodia and dendrite-like structures in other cell types and is activated by several different hormonally regulated second messenger systems. Based on our initial studies, we hypothesize that rac1 is a central convergence point for the protein kinase A (PKA), protein kinase C (PKC) and Ca2+ second messenger systems, and that melanosome transfer to keratinocytes is closely linked to activation of rac1. Further, we hypothesize that melanosome transfer is regulated through PKA- dependent phosphorylation of melanosomal associated proteins and that rab3a and rabphilin, proteins involved in regulated exocytosis in other cell types, are regulatory molecules for melanosome transfer to keratinocytes. The studies proposed in this proposal are the first to focus on and target these two processes in a mammalian model using state of the art molecular and cell biology technqiues, and results from this work would represent a major advance in a field which has been largely confined to morphologic analyses. Results from these studies could have significant implications for the design of new therapies to enhance the ability of melanocytes to provide photoprotection to the skin, and thus decrease the incidence of cutaneous cancer.
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