Melanosome transfer, photoreception and toxicity assays in melanophores

Many animals such as fish and frogs have developed the ability to change colour of their skin to adapt to the environment or to signal to other individuals. This ability is due to specialised skin cells called melanophores. Melanophores contain thousands of melanosomes, small membrane-enclosed organelles containing the black or brown pigment melanin. The melanosomes can aggregate to the cell centre rendering the cells pale or disperse throughout the cell to become dark. The intracellular transport of melanosomes is regulated by neuronal or hormonal external stimuli. Fast colour change is achieved by aggregation/dispersion of melanosomes but long-term colour change can also be achieved by melanosome transfer to surrounding skin cells. An amphibian immortalized melanophore cell line was used from the African claw frog, Xenopus laevis to study transfer of melanosomes to co-cultured fibroblasts. Melanosome transfer was observed and up regulated by the hormone α-MSH . The transfer was quantified using light-, fluorescence and electron microscopy. A new and powerful method for transfer experiments was developed. Fluorescent semiconductor nanocrystals, qdots, were used in combination with flow cytometry. The qdots were taken up by the cultured Xenopus laevis melanophores, localised to the melanosomes and transferred to co-cultured fibroblasts. The method is a step towards enabling large scale analysis of pigment transfer. Xenopus laevis melanophores can be cultivated in 96-well culture plates which allow quantification of aggregation or dispersion in a fast and reproductive way. Glyphosate containing herbicides, i.e. Roundup, are commonly used in the world, but some toxic effects have been found on amphibians in vivo and human and mouse cells in vitro. To learn more about potential effects on intracellular transport and the cytoskeleton in animal Roundup, glyphosate, glyphosateisopropylamine and isopropylamine were tested on the transport of melanosomes to the cell centre by spectrophotmetry and by fluorescence microscopy on microtubules and actin filaments. All tested compounds inhibited the aggregation and affected the morphology of the cytoskeleton. The effect was found to be pH dependent. Amphibian melanophores can be regulated directly by light via a melanopsin receptor. Photoreception was found in cultured early embryos of the zebrafish Danio rerio. Light induced dispersion of the melanophores was contrast to what is found at adults when light causes aggregation of the melanosomes due to signals from the CNS. At least one subclass of melanopsin was detected in the zebrafish retinal pigment epithelial cells

Dermal papilla cells and melanocytes response to physiological oxygen levels depends on their interactions

Background: Human dermal papilla (DP) cells and melanocytes (hMel) are central players in hair growth and pigmentation, respectively. In hair follicles (HFs), oxygen (O2) levels average 5%, being coupled with the production of reactive oxygen species (ROS), necessary to promote hair growth.Materials and Methods: DP cell and hMel proliferation and phenotype were stud-ied under physiological (5%O2, physoxia) or atmospheric (21%O2, normoxia) oxygen levels. hMel-DP cells interactions were studied in indirect co-culture or by directly co-culturing hMel with DP spheroids, to test whether their interaction affected the response to physoxia.Results: Physoxia decreased DP cell senescence and improved their secretome and phenotype, as well as hMel proliferation, migration, and tyrosinase activity. In indi-rect co- cultures, physoxia affected DP cellsâ alkaline phosphatase (ALP) activity but their signalling did not influence hMel proliferation or tyrosinase activity. Additionally, ROS production was higher than in monocultures but a direct correlation between ROS generation and ALP activity in DP cells was not observed. In the 3D aggregates, where hMel are organized around the DP, both hMel tyrosinase and DP cells ALP ac-tivities, their main functional indicators, plus ROS production were higher in physoxia than normoxia.Conclusions: Overall, we showed that the response to physoxia differs according to hMel-DP cells interactions and that the microenvironment recreated when in direct contact favours their functions, which can be relevant for hair regeneration purposes.We thank Dr Luca Gasperini for his technical assistance in the image analysis using the CellProfiler™ software. The authors also thank the financial support given by the European Research Council through the consolidator grant “ECM_INK” (ERC-2016-COG-726061) and by FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) through the PD/59/2013, PD/BD/113800/2015 (C. Abreu) and IF/00945/2014 (A. P. Marques) grants