Title: Melanin Transfer: The Keratinocytes Are More than Gluttons
Abstract: Skin pigmentation is tightly linked to the transfer of melanin from melanocytes to neighboring keratinocytes. For decades, cellular mechanisms underlying pigment transfer have remained enigmatic. Tarafder et al. identify a keratinocyte-initiated process coupling the exocytosis and endocytosis of melanin as a major pigment transfer mode in epidermis. These findings open new paths in our understanding of melanocyte–keratinocyte communication regulating pigmentation. Skin pigmentation is tightly linked to the transfer of melanin from melanocytes to neighboring keratinocytes. For decades, cellular mechanisms underlying pigment transfer have remained enigmatic. Tarafder et al. identify a keratinocyte-initiated process coupling the exocytosis and endocytosis of melanin as a major pigment transfer mode in epidermis. These findings open new paths in our understanding of melanocyte–keratinocyte communication regulating pigmentation. The epidermis, the outer layer of skin, consists mainly of two cell types, keratinocytes and melanocytes. Melanocytes synthesize and store melanin pigment within membrane-enclosed lysosome-related organelles (LROs) called melanosomes. However, the pigment that one sees in skin is primarily within keratinocytes, the recipient cells for melanin. Transfer of pigment occurs within epidermal melanin units in which melanocytes extend long dendrites that contact up to 40 keratinocytes. This mechanism suggests that pigment transfer must be efficient and tightly regulated in order to provide full photo-protection. Early electron microscopy studies on keratinocytes revealed that melanin cores were surrounded by one or two membranes. These observations led to non-mutually exclusive hypotheses for the mechanism of melanin transfer: (1) (cyto)phagocytosis of melanocyte dendrites (filopodia) by keratinocytes; (2) melanosome exocytosis of “naked melanin” (melanocore) into the extracellular space, followed by internalization by keratinocytes; (3) melanosome transport via membrane nanotubes; and/or (4) shedding by melanocytes of melanosome-enriched structures that are then phagocytosed by keratinocytes (Van Den Bossche and Naeyaert, 2006Van Den Bossche K. Naeyaert J.M. et al.The quest for the mechanism of melanin transfer.Traffic. 2006; 7: 769-778Crossref PubMed Scopus (185) Google Scholar). These hypothetical modes of transfer had been deduced from observations in different model systems, ranging from studies of human and animal skin to the use of in vitro co-cultures of melanocytes and keratinocytes procured from diverse species and/or phototypes. One may postulate that these different cellular processes co-exist,depending on the skin areas or phototypes and UV exposure, even if some are likely to be more preponderant than others. Characterizing the mechanisms involved in melanin transfer has important clinical consequences for pigmentary disorders, photocarcinogenesis and pigment- and/or melanosome-dependent resistance of melanomas to chemotherapies (Chen et al., 2006Chen K.G. Valencia J.C. Lai B. et al.Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas.Proc Natl Acad Sci USA. 2006; 103: 9903-9907Crossref PubMed Scopus (143) Google Scholar). In this issue, Tarafder et al., 2014Tarafder A.K. Bolasco G. Correia M.S. et al.Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.J Invest Dermatol. 2014; 134: 1056-1066Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar use electron microscopy to characterize pigment transfer. They present several lines of evidence suggesting that one major mode of transfer is exocytosis of polymerized melanin, referred to as melanocores, by fusion of melanosomes with the melanocyte plasma membrane, followed by internalization by keratinocytes at sites of close melanocyte/keratinocyte contact. Although this is not a completely new concept, the authors provide important new support for this mode, using human skin samples that are either chemically fixed or immobilized quickly by high-pressure freezing, allowing optimal tissue preservation. Moreover and in contrast to other studies, they perform serial sectioning of their samples, allowing them to follow continuity of the same structure of interest over several micrometers in the epidermal pigment unit. Exploiting these methods, they identify melanin alone, without any surrounding membrane, in the extracellular space, suggesting that the melanosomes had fused with the melanocyte plasma membranes, followed by release. The melanocore was then fated for internalization by keratinocytes. Although such an event is quite dynamic, uptake of the melanocore by keratinocytes was occasionally observed, supporting the interpretation that keratinocytes ingest the “great dark meal”. Then, Tarafder et al., 2014Tarafder A.K. Bolasco G. Correia M.S. et al.Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.J Invest Dermatol. 2014; 134: 1056-1066Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar analyzed the fate of pigment within the keratinocyte. They demonstrated that the melanocore is surrounded by one membrane that lacks TYRP1, a protein that is generally present on the limiting melanosomal membrane within melanocytes (Raposo et al., 2001Raposo G. Tenza D. Murphy D.M. et al.Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.J Cell Biol. 2001; 152: 809-824Crossref PubMed Scopus (357) Google Scholar), suggesting that the limiting membrane was not derived from the melanocyte but that it originated from the keratinocyte. Although other transfer hypotheses cannot be excluded, based on the fact that they were not observed, the data nevertheless suggest that one major mode in human epidermis is coupled exo/endocytosis of the pigment. Keratinocytes control many aspects of the biology of melanocytes, including expression of enzymes that synthesize melanin and transporters, biogenesis and transport of melanosomes (Yamaguchi and Hearing, 2009Yamaguchi Y. Hearing V.J. Physiological factors that regulate skin pigmentation.Biofactors. 2009; 35: 193-199Crossref PubMed Scopus (351) Google Scholar), melanocyte recruitment to the epidermal melanin unit, (Weiner et al., 2007Weiner L. Han R. Scicchitano B.M. et al.Dedicated epithelial recipient cells determine pigmentation patterns.Cell. 2007; 130: 932-942Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and pigment transfer (Cardinali et al., 2008Cardinali G. Bolasco G. Aspite N. et al.Melanosome transfer promoted by keratinocyte growth factor in light and dark skin-derived keratinocytes.J Invest Dermatol. 2008; 128: 558-567Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The authors designed a cell culture melanin exocytosis assay to investigate quantitatively the role of keratinocytes in this process. Using this approach, they reinforced the idea that keratinocytes induce the exocytosis of melanocores by showing enhanced pigment secretion by melanocytes in the presence of keratinocytes, but not irrelevant cells such as fibroblasts. Tarafder et al., 2014Tarafder A.K. Bolasco G. Correia M.S. et al.Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.J Invest Dermatol. 2014; 134: 1056-1066Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar also addressed the molecular factors that control pigment exocytosis and whether keratinocytes can modulate this pathway. Master regulators of cargo delivery and organelle transport are the family of small Rabs GTPases that recruit effector proteins to membranes that, in turn, control several intracellular trafficking events. In melanocytes, several RABs were shown to modulate pigmentation by controlling melanosome biogenesis (RAB32/38) (Wasmeier et al., 2006Wasmeier C. Romao M. Plowright L. et al.Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes.J Cell Biol. 2006; 175: 271-281Crossref PubMed Scopus (212) Google Scholar), peripheral transport and tethering of melanosomes (RAB27A), and intracellular melanin content (RAB11/17) (Beaumont et al., 2011Beaumont K.A. Hamilton N.A. Moores M.T. et al.The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.Traffic. 2011; 12: 627-643Crossref PubMed Scopus (68) Google Scholar; Marks et al., 2013Marks M.S. Heijnen H.F. Raposo G. Lysosome-related organelles: unusual compartments become mainstream.Curr Opin Cell Biol. 2013; 25: 495-505Crossref PubMed Scopus (192) Google Scholar). By depleting or overexpressing RAB proteins in primary or immortalized melanocytes, Tarafder et al., 2014Tarafder A.K. Bolasco G. Correia M.S. et al.Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.J Invest Dermatol. 2014; 134: 1056-1066Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar demonstrated that RAB11B, but not RAB27A or RAB11A, controls keratinocyte-induced exocytosis of the pigment. These findings are in agreement with previous studies showing that RAB11B-inactivated melanocytes increase their intracellular melanin content (Beaumont et al., 2011Beaumont K.A. Hamilton N.A. Moores M.T. et al.The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.Traffic. 2011; 12: 627-643Crossref PubMed Scopus (68) Google Scholar). This result suggests a fascinating process in which keratinocytes modulate the activity of intracellular trafficking machineries required for efficient pigment transfer. At this point, it would be useful to understand RAB11B-dependent melanosome exocytosis at a molecular level. RAB11B is an ubiquitously expressed member of the RAB11 family, which controls classical recycling functions as well as specific recycling and exocytosis events in neurons or polarized epithelial cells. Melanocytes are highly specialized cells that elaborate a complex recycling endosomal network in order to generate and maintain pigmented melanosomes (Delevoye et al., 2009Delevoye C. Hurbain I. Tenza D. et al.AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.J Cell Biol. 2009; 187: 247-264Crossref PubMed Scopus (130) Google Scholar). The study by Tarafder et al., 2014Tarafder A.K. Bolasco G. Correia M.S. et al.Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.J Invest Dermatol. 2014; 134: 1056-1066Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar suggests that a RAB11B-positive endosomal subdomain contributes to the pigment exocytosis required for melanin transfer, whereas other related recycling domains sustain melanin synthesis (Delevoye et al., 2009Delevoye C. Hurbain I. Tenza D. et al.AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.J Cell Biol. 2009; 187: 247-264Crossref PubMed Scopus (130) Google Scholar). This suggests that several subdomains of recycling endosomes might sequentially modulate the biogenesis of melanosome and its secretion, once fully mature. Although speculative keratinocytes may control pigment exocytosis by activating melanocyte-dependent signaling pathways via secreted soluble factors and/or by direct contact. Interestingly, RAB11-positive vesicles have been involved in the inducible secretion of an another LRO, the cytotoxic granule of cytotoxic T lymphocytes. It is intriguing that specialized cell types share molecular machineries required for both LRO biogenesis and secretion (Marks et al., 2013Marks M.S. Heijnen H.F. Raposo G. Lysosome-related organelles: unusual compartments become mainstream.Curr Opin Cell Biol. 2013; 25: 495-505Crossref PubMed Scopus (192) Google Scholar). Many questions can now be addressed. For instance, the authors confirm previous findings showing that RAB11-positive endosomes are constitutively apposed to pigmented melanosomes (Delevoye et al., 2009Delevoye C. Hurbain I. Tenza D. et al.AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.J Cell Biol. 2009; 187: 247-264Crossref PubMed Scopus (130) Google Scholar). It would be of interest to define the distribution of recycling endosomal subdomains (RAB11B/ RAB17) and melanosomes under stimulated conditions (co-culture, UV irradiation). Several other questions can also be addressed. Do recycling endosomes fuse with melanosomes before exocytosis, as during cytotoxic granule secretion, and/or are they involved in the delivery of the melanosomal components required for plasma membrane fusion? How do keratinocytes have an impact on intracellular trafficking within melanocytes, and how do they have an impact on melanin exocytosis? Do keratinocytes need to contact melanocytes physically in order to signal, as predicted by their close appositions within the epidermal melanin unit, and is the pigment transfer process similar in all skin phototypes? Wu et al., 2012Wu X.S. Masedunskas A. Weigert R. et al.Melanoregulin regulates a shedding mechanism that drives melanosome transfer from melanocytes to keratinocytes.Proc Natl Acad Sci USA. 2012; 109: E2101-E2109Crossref PubMed Scopus (62) Google Scholar showed recently that pigment transfer in the skin of mouse ear and in cell culture occurs via the shedding of pigment-enriched regions and their subsequent phagocytosis by keratinocytes. In this issue, Tarafder et al., 2014Tarafder A.K. Bolasco G. Correia M.S. et al.Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.J Invest Dermatol. 2014; 134: 1056-1066Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar identify “naked” melanin in the extracellular space of human skin samples, and they report that keratinocytes “fine–tune” the exocytosis of melanin by melanocytes before its uptake. Both sets of observations are valuable, and they advance our understanding of the mechanisms that control pigment transfer. But why differences are observed is not clear. Therefore, further studies are needed to decipher one of the main secrets kept in the pigmentation field. I thank all members of the Structure and Membrane Compartment group, especially Graça Raposo, Ilse Hurbain, and Léa Ripoll for helpful comments.