title: Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.
abstract: Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV) stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs) and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1) localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin's physiological response to UV radiation.
authors.affiliation: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island, United States of America.
authors.forename: Jan M
authors.initials: JM
authors.lastname: Bruder
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Pfeiffer
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Ciriello
authors.forename: Diana M
authors.initials: DM
authors.lastname: Horrigan
authors.forename: Nadine L
authors.initials: NL
authors.lastname: Wicks
authors.forename: Benjamin
authors.initials: B
authors.lastname: Flaherty
authors.forename: Elena
authors.initials: E
authors.lastname: Oancea
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000528:radiation effects; D018920:Coculture Techniques; D004058:Diffusion; D004307:Dose-Response Relationship, Radiation; D005136:Eye Proteins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D008546:Melanoma, Experimental / Q000473:pathology; D020460:Melanosomes / Q000378:metabolism* / Q000528:radiation effects; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D057054:Molecular Imaging; D009068:Movement / Q000528:radiation effects; D021381:Protein Transport / Q000528:radiation effects; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D014466:Ultraviolet Rays; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
chemical_list: D005136:Eye Proteins; D005456:Fluorescent Dyes; C506821:Gpr143 protein, mouse; D008562:Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D000075745:rab27 GTP-Binding Proteins; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins