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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

Funding: We thank Liliana Bento for valuable assistance in formatting the manuscript. This article was supported by the LYSOCIL project. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 811087. CGA has funding from Maratona da Saúde 2016 and Fundaçao para a Ciência e a Tecnologia ˜ , I.P. (CEECIND/00410/2017). FMP is a Wellcome Investigator in Science and a Royal Society Wilson merit award holder. Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has revealed that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling, and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyse lysosome morphology, positioning, motility, and function. We highlight the principles behind these methods, the methodological strategies, and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists. publishersversion published

Funding Information: Funding: This work was supported by Fundação para a Ciência e Tecnologia project PTDC/MED-PAT/30385/2017 and iNOVA4Health-UIDB/04462/2020, a program financially supported by Fun-dação para a Ciência e Tecnologia/Ministério da Educação e Ciência, Portugal, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. Funding Information: This work was supported by Fundação para a Ciência e Tecnologia project PTDC/MEDPAT/30385/2017 and iNOVA4Health-UIDB/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, Portugal, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Early and intermediate AMD patients represent a heterogeneous population with an important but variable risk of progression to more advanced stages of the disease. The five-year progression from early and intermediate AMD to late disease is known to range from 0.4% to 53%. This wide variation explains the particular interest in searching predictive AMD biomarkers. Clinical parameters such as drusen size, presence of pigmentary abnormalities, and fellow eye status were, traditionally, the more important predictive elements. Multimodal retinal assessment (Color Fundus Photography, Optical Coherence Tomography, Optical Coherence Angiography and Fundus Autofluorescence) is providing new and accurate image biomarkers, useful in research and in daily practice. If individual progression risk could be anticipated, then management plans should be adapted accordingly, considering follow-up intervals and therapeutic interventions. Here, we reviewed the most important image progression biomarkers of early and intermediate AMD with relevant interest in clinical practice. publishersversion published