Tese de Doutoramento Biomedicina 2022 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Skin pigmentation is essential for protection against ultra-violet radiation-(UVr)-induced damage. This photoprotection is sustained by the pigment melanin and requires the crosstalk between two cell types present in the skin epidermis: melanocytes and keratinocytes. Melanin is synthesized and stored within melanosomes, a type of lysosome-related organelle (LRO) present in melanocytes and transferred to surrounding keratinocytes. Within keratinocytes, melanin accumulates above the nuclei shielding the DNA from UVr. Although melanin synthesis and melanosome biogenesis have been extensively characterized, the mechanisms regulating melanosome exocytosis are poorly understood. Moreover, LROs and lysosomes share several common features, including the biogenesis pathway, low intraluminal pH, and the presence of hydrolytic enzymes and lysosomal membrane proteins. However, the capacity to undergo regulated exocytosis was thought to be exclusive to LROs, whereas lysosomes were considered as the end-point of the endocytic pathway. Nevertheless, lysosome exocytosis is now regarded as an ubiquitous mechanism present in most cell types, essential for their survival. While searching for lysosome and melanosome exocytosis regulators we found that these exocytic processes require the coordinated function of both Rab11 and Rab3a small GTPases. We observed that lysosome exocytosis is regulated by a Rab11-Rab3a cascade, required for the delivery of the Rab3a guanine nucleotide exchange factor GRAB, essential for the activation of Rab3a on lysosomes before secretion. In contrast, Rab11b and Rab3a appear to have different functions in melanocytes, as we found evidence for two distinct exocytic pathways: a non-stimulated Rab11b-dependent pathway and a keratinocyte-conditioned media (KCM)-stimulated Rab3a-dependent pathway. Additionally, we were also interested in uncovering the mechanisms regulating melanin endocytosis by keratinocytes. We previously found evidence that the predominant model of melanin transfer is exo/phagocytosis of melanocores. By comparing membraneless melanocores and melanosomes, we found that distinct Rho GTPases are required for their internalization by keratinocytes. Furthermore, we collected evidence suggesting the protease-activated receptor 2-dependent phagocytosis of melanocores, whereas we observed that melanosomes enter keratinocytes through macropinocytosis. Thus, we conclude that the form of melanin presented to keratinocytes directly influences the internalization route followed. Finally, we investigated the mechanisms regulating melanin processing by keratinocytes. Previously, we have reported that melanin accumulates in non-degradative compartments. Here, we showed that melanin interacts/fuses with lysosomes early after internalization. Our results suggest that this interaction/fusion is essential for the formation of a non-degradative melanin storage compartment within keratinocytes, which we named melanokerasome. We also explored the role of autophagy in melanin processing, since it has been established as a regulator of melanin degradation inside keratinocytes. Interestingly, we found that melanocores, but not melanosomes, interfere with the autophagic activity of keratinocytes, possibly by decreasing autophagosome biogenesis. Finally, our results suggest that autophagy reactivation results in partial melanin degradation and can explain the formation of melanin clusters in light skins. These studies provide evidence for shared regulators of lysosome and melanosome exocytosis, which can be exploited in cases where the secretion of both organelles is affected, such as in melanoma. Moreover, these studies contribute to a better understanding of the mechanisms regulating melanin internalization and processing by keratinocytes, processes that have been less explored in the field of skin pigmentation. The discoveries made are essential for the understanding of the complex mechanisms involved in skin pigmentation. This will be particularly important for the development of new therapeutic and cosmetic strategies that can be applied to increase photoprotection, revert hypo and hyperpigmented lesions, as well as to understand the pathophysiology of more complex pigmentary disorders