Search

Funding: The authors would like to thank the scientific and technical assistance from the CEDOC Cell Culture, Flow Cytometry and Microscopy facilities. We also thank the Electron Microscopy Facility of Instituto Gulbenkian de Ciência for technical assistance. The authors also thank Dorothy Bennett and Elena Sviderskaya (St. George's University of London, UK), Susana Lopes (CEDOC, NOVA Medical School) and Paulo Matos (National Health Institute Doutor Ricardo Jorge, Portugal) for the kind gift of reagents and Paulo Matos also for expert advice. This article was supported by the LYSOCIL project. This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 811087. This article was also supported by Fundaçao para a Ciência e a Tecnologia ˜ (FCT), Portugal through grant PTDC/BIA-CEL/29765/2017, PhD fellowships to HM, MVN, LBL and LCC (PD/BD/114118/2015, PD/BD/137442/2018, SFRH/BD/131938/2017 and 2020.8812.BD, respectively), the FCT Investigator Program to DCB (IF/00501/2014/ CP1252/CT0001), and FCT Unit iNOVA4Health – UIDB/04462/2020 and UIDP/04462/2020, a programme financially supported by FCT / Ministério da Ciência, Tecnologia e Ensino Superior, through national funds. In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred - either in a membrane-bound melanosome or as a melanosome core, i.e., melanocore. Here, we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures, with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1, CtBP1/BARS, Cdc42 or RhoA, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Interestingly, we found that Rac1, Cdc42 and RhoA are differently activated by melanocore or melanosome stimulation, supporting the existence of two distinct internalization routes of melanin internalization. Furthermore, we show that melanocore uptake induces Protease-activated receptor-2 (PAR-2) internalization by keratinocytes to a higher extent than melanosomes. Since skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore-based mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis. publishersversion published