Biogenesis of melanosomes in Kupffer cells of Proteus anguinus (Urodela,Amphibia)

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single-membrane-bounded bodies, named as 'clusters of melanosomes' or 'melanosomogenesis centers'. Inside such clusters, different structures are present: (1) filament-like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron-dense melanosomes in different stages of melanization, (3) melanosomes with an electron-dense cortical area and a less electron-dense medullar area, and (4) uniformly highly electron-dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.     

Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron‐induced peroxidation of lipids

Melanin in the human retinal pigment epithelium (RPE) is believed to play an important photoprotective role. However, unlike in skin, melanosomes in the RPE are rather long‐lived organelles, which increases their risk of modifications resulting from significant fluxes of light and high oxygen tension. In this work, we subjected purified bovine RPE melanosomes to prolonged aerobic exposure with intense visible and near ultraviolet radiation and studied the effects of irradiation on the melanosome's capacity to inhibit peroxidation of lipids induced by iron/ascorbate. We found that control, untreated melanosomes show a concentration‐dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolysed melanosomes lose their antioxidant efficiency and even became prooxidant. The prooxidant action of partially photobleached melanosomes was observed for pigment granules with a melanin content reduced by about 50% compared with untreated melanosomes, as determined by electron spin resonance spectroscopy. We have previously shown that a similar loss in the content of the RPE melanin occurs during human lifetime, which may suggest that the normal antioxidant properties of human RPE melanin become compromised with aging.     

Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron-induced peroxidation of lipids

Melanin in the human retinal pigment epithelium (RPE) is believed to play an important photoprotective role. However, unlike in skin, melanosomes in the RPE are rather long-lived organelles, which increases their risk of modifications resulting from significant fluxes of light and high oxygen tension. In this work, we subjected purified bovine RPE melanosomes to prolonged aerobic exposure with intense visible and near ultraviolet radiation and studied the effects of irradiation on the melanosome's capacity to inhibit peroxidation of lipids induced by iron/ascorbate. We found that control, untreated melanosomes show a concentration-dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolysed melanosomes lose their antioxidant efficiency and even became prooxidant. The prooxidant action of partially photobleached melanosomes was observed for pigment granules with a melanin content reduced by about 50% compared with untreated melanosomes, as determined by electron spin resonance spectroscopy. We have previously shown that a similar loss in the content of the RPE melanin occurs during human lifetime, which may suggest that the normal antioxidant properties of human RPE melanin become compromised with aging.     

Biogenesis of Melanosomes in Kupffer Cells of Proteus anguinus (Urodela,Amphibia)

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single‐membrane‐bounded bodies, named as ‘clusters of melanosomes’ or ‘melanosomogenesis centers’. Inside such clusters, different structures are present: (1) filament‐like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron‐dense melanosomes in different stages of melanization, (3) melanosomes with an electron‐dense cortical area and a less electron‐dense medullar area, and (4) uniformly highly electron‐dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.     

Morphological and geochemical evidence of eumelanin preservation in the feathers of the Early Cretaceous bird, Gansus yumenensis

Recent studies have shown evidence for the preservation of colour in fossilized soft tissues by imaging melanosomes, melanin pigment containing organelles. This study combines geochemical analyses with morphological observations to investigate the preservation of melanosomes and melanin within feathers of the Early Cretaceous bird, Gansus yumenensis. Scanning electron microscopy reveals structures concordant with those previously identified as eumelanosomes within visually dark areas of the feathers but not in lighter areas or sedimentary matrices. Fourier transform infrared analyses show different spectra for the feathers and their matrices; melanic functional groups appear in the feather including carboxylic acid and ketone groups that are not seen in the matrix. When mapped, the carboxylic acid group absorption faithfully replicates the visually dark areas of the feathers. Electron Paramagnetic Resonance spectroscopy of one specimen demonstrates the presence of organic signals but proved too insensitive to resolve melanin. Pyrolysis gas chromatography mass spectrometry shows a similar distribution of aliphatic material within both feathers that are different from those of their respective matrices. In combination, these techniques strongly suggest that not only do the feathers contain endogenous organic material, but that both geochemical and morphological evidence supports the preservation of original eumelanic pigment residue.     

Excess tyrosine restores the morphology and maturation of melanosomes affected by the murine slaty mutation

The slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase in melanocytes and to reduce the melanin content in the skin, hairs, and eyes. The slaty gene is known to be important for maximizing melanin deposition in melanosomes. However, it was not known whether the slaty mutation affects the morphology of melanosomes. Moreover, it was unknown whether melanosome development is modulated by melanogenic factors. In this study, the characteristics of melanosomes of slaty melanocytes in serum-free primary culture were investigated in detail under the electron microscope. In slaty melanocytes, melanosome maturation was blocked at stage III, and numerous spherical melanosomes with globular depositions of pigment in addition to elliptical melanosomes were observed. L-tyrosine (Tyr), the starting material of melanin synthesis, is known to stimulate melanin synthesis. To clarify whether L-Tyr restores the reduced production of melanin, L-Tyr was added to the culture medium and tested for its melanogenic effect. L-Tyr greatly increased the number and percentage of mature stage IV melanosomes. Moreover, L-Tyr increased elliptical melanosomes, but decreased spherical melanosomes. These results suggest that the slaty mutation inhibits the development of elliptical stage IV melanosomes, and that L-Tyr restores the development of elliptical stage IV melanosomes. L-Tyr seems to restore both the morphology and maturation of melanosomes affected by the slaty mutation.     

Current challenges in understanding melanogenesis: bridging chemistry,biological control,morphology, and function

Melanin is a natural pigment produced within organelles, melanosomes, located in melanocytes. Biological functions of melanosomes are often attributed to the unique chemical properties of the melanins they contain; however, the molecular structure of melanins, the mechanism by which the pigment is produced, and how the pigment is organized within the melanosome remains to be fully understood. In this review, we examine the current understanding of the initial chemical steps in the melanogenesis. Most natural melanins are mixtures of eumelanin and pheomelanin, and so after presenting the current understanding of the individual pigments, we focus on the mixed melanin systems, with a critical eye towards understanding how studies on individual melanin do and do not provide insight in the molecular aspects of their structures. We conclude the review with a discussion of important issues that must be addressed in future research efforts to more fully understand the relationship between molecular and functional properties of this important class of natural pigments.     

Different approaches for assaying melanosome transfer

Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report, we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP-labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably because of incorrect sorting or deficient incorporation of the fluorescent protein and different localization. We were able to label melanosomes via incorporation of 14C-thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co-culture. Differential trypsinization and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer.     

Incorporation and binding of estrogens into melanin: comparison of mushroom and mammalian tyrosinases.

The activities of mushroom and melanoma tyrosinases towards the estrogens were compared. While the fungal enzyme is capable of hydroxylating estradiol to the 2-hydroxy compound and to oxidize the latter to the quinone, the mammalian enzyme does not have this ability. With dopa as substrate and an estrogen present in the reaction mixture, both enzyme reactions yield melanin with the steroid firmly incorporated into the pigment, although with the mammalian enzyme the incorporation is small. The steroid appears to be incorporated by covalent linkage. It is suggested that the incorporation of estrogens into melanin produced by mammalian tyrosinase is via their oxidation by oxidized intermediates of the dopa to melanin transformation. Melanin itself may function as oxidant for the estrogens. Whole melanoma cells are capable of binding estrogens and incorporating small amounts into melanosomes. Similarly, fresh melanosomes in isolation can incorporate estrogens into their structure, presumably by covalent bonding to their melanin.     

Regulation of Melanogenesis in Melanocytes

Melanogenesis refers to the biosynthesis of melanin pigment in pigment cells called melanocytes. Melanins are mixed biopolymers formed during a series of oxidation/reduction reactions that are initiated by the enzymatic hydroxylation of L-tyrosine to L-dopa. In living cells, melanogenesis is limited to melanosomes, the membrane bounded microscopic secretory granules of melanocytes. Melanosomes may be secreted into the environment as, for example, from the squid's ink gland; or be transferred to neighboring cells, such as the keratinocytes in human skin and hair; or they may remain within the pigment cell and change only their subcellular localization, as in the rapidly color-changing dermis of lower vertebrates. Regulation of the melanocytic phenotype involves synthesis of the biosynthetically active subcellular apparatus of melanogenesis, premelanosomes and tyrosinase, and the utilization of the final product, melanized melanosomes, in the translocation and secretory processes mentioned above. Genetic information for this regulation is stored in the nuclear genome whose expression is controlled by the intra- and extracellular environment. As premelanosomes become biosynthetically active, they mature into melanosomes by fusing with vesicles derived from the trans-Golgi network and the plasmalemma, thereby internalizing and incorporating contents and membrane components from inside the cell and the cell surface. In the process, melanosomes become acidified. The thesis pursued in this review explores the importance of the melanosome as the final common pathway of regulation of melanin biosynthesis.     

Melanocytes: the new Black

Melanocytes, pigment-producing cells residing primarily in the hair follicle, epidermis and eye, are responsible for skin hair and eye pigmentation. Pigmentation is achieved by the highly regulated manufacture of the pigment melanin in specialised organelles, melanosomes that are transported along dendritic processes before being transferred to growing hair, or keratinocytes where melanin protects from UV-induced DNA damage. Because loss of melanocytes gives a clear pigmentation phenotype yet is non-lethal, over 130 genes implicated in the development or function of this cell type have been identified to date, and in humans the loss of melanocytes or their ability to produce pigment, or transport or transfer melanosomes is associated with several diseases such as vitiligo, albinism and Hermansky-Pudlak syndrome. Importantly, the effective combination of genetics, cell and molecular biology possible with this cell type is attracting an increasing number of researchers focussed on understanding how cells coordinate survival, proliferation, differentiation and stem cell maintenance.     

Interpreting melanin-based coloration through deep time: a critical review

Colour, derived primarily from melanin and/or carotenoid pigments, is integral to many aspects of behaviour in living vertebrates, including social signalling, sexual display and crypsis. Thus, identifying biochromes in extinct animals can shed light on the acquisition and evolution of these biological traits. Both eumelanin and melanin-containing cellular organelles (melanosomes) are preserved in fossils, but recognizing traces of ancient melanin-based coloration is fraught with interpretative ambiguity, especially when observations are based on morphological evidence alone. Assigning microbodies (or, more often reported, their ‘mouldic impressions’) as melanosome traces without adequately excluding a bacterial origin is also problematic because microbes are pervasive and intimately involved in organismal degradation. Additionally, some forms synthesize melanin. In this review, we survey both vertebrate and microbial melanization, and explore the conflicts influencing assessment of microbodies preserved in association with ancient animal soft tissues. We discuss the types of data used to interpret fossil melanosomes and evaluate whether these are sufficient for definitive diagnosis. Finally, we outline an integrated morphological and geochemical approach for detecting endogenous pigment remains and associated microstructures in multimillion-year-old fossils.     

Effect of the barring gene on eye pigmentation in the fowl

Pigment cells of the iris, pecten, retinal pigment epithelium, and choroid of the wild-type jungle fowl (JF) and the barred Plymouth rock (BPR) breeds of adult chickens were studied at both light and electron microscopic levels. BPR choroidal tissues had 2.8 times fewer melanophores than the JF choroid, and BPR melanophores also contained 2.4 times fewer melanosomes, which tended to clump together in variously sized clusters. The melanosomes were often irregular in shape, smaller in diameter, and less mature (stage III) than those granules in the JF. The retinal pigment epithelium of both JF and BPR breeds contained a single epithelial layer of columnar cells. Rod-shaped melanosomes were present in the more apical regions of this cell type in both breeds. Both JF and BPR irides contained a multilayered posterior pigmented epithelium of columnar shaped cells that were densely filled with large spherical granules. Intercellular spaces with interdigitating cytoplasmic projections were present between pigment cells of both breeds. The pecten melanophores of both breeds were dendritic with melanosomes that were larger and fewer in numbers than those pigment cells of the iris and choroid. Intercellular spaces were present between cells in both breeds, with numerous villous-like pigment cell extensions. Choroid melanophores contained very little, if any, acid phosphatase activity. Approximately one-half of the retinal pigment epithelial cells observed contained small amounts of diffuse acid phosphatase activity in both breeds. The iris and pecten melanophores of both breeds contained profuse acid phosphatase activity scattered throughout their cytoplasms. Sparse tyrosinase activity was seen in iris and pecten pigment cells, whereas no tyrosine activity was observed in choroid melanophores or in retinal pigment epithelial cells in the two breeds, indicating that little new melanogenesis occurs in adult pigmented eye tissues. The results show that the barring gene reduces the number and melanin content of the choroidal melanophores in homozygous male BPR chickens as compared to the wild-type JF chickens. Whether this gene prevents the initial migration of embryonic neural crest cells (future melanophores) to the choroid or whether some of the choroidal melanophores prematurely degenerate in the embryo of young birds is yet to be determined. If the latter is the case, this choroid system may serve as a model for a genetic hypomelanotic disease such as vitiligo.     

The antimicrobial properties of melanocytes, melanosomes and melanin and the evolution of black skin.

A biological issue that has not been satisfactorily resolved is the role of melanin in skin and other animal tissues. A hypothesis is outlined here to account for the evolution of black skin and the ubiquity of melanin in vertebrate tissues. Evidence is presented that melanization of skin and other tissues forms an important component of the innate immune defense system. A major function of melanocytes, melanosomes and melanin in skin is to inhibit the proliferation of bacterial, fungal and other parasitic infections of the dermis and epidermis. This function can potentially explain (a) the latitudinal gradient in melanization of human skin; (b) the fact that melanocyte and melanization patterns among different parts of the vertebrate body do not reflect exposure to radiation; (c) provide a theoretical framework for recent empirical findings concerning the antimicrobial activity of melanocytes and melanosomes and their regulation by known mediators of inflammatory responses.     

A newly discovered pathway of melanin formation in cultured retinal pigment epithelium of cattle

According to a recent hypothesis the melanin granules in the retinal pigment epithelium of mammals originate from photosensory membrane degradation. To test this hypothesis the retinal pigment epithelium of cattle was kept in tissue culture and exposed to gold-labelled rod outer segments. Gold granules were later detected inside phagosomes, melanosomes and mature melanin granules. Tyrosinase, the key enzyme in melanogenesis, was additionally localized inside phagosomes. These results indicate that in cultured retinal pigment epithelium the matrix of the melanosome can originate from phagosomes. therefore, the melanosome is a specialized lysosome.     

Synchrotron x‐ray fluorescence analysis reveals diagenetic alteration of fossil melanosome trace metal chemistry

A key feature of the pigment melanin is its high binding affinity for trace metal ions. In modern vertebrates trace metals associated with melanosomes, melanin‐rich organelles, can show tissue‐specific and taxon‐specific distribution patterns. Such signals preserve in fossil melanosomes, informing on the anatomy and phylogenetic affinities of fossil vertebrates. Fossil and modern melanosomes, however, often differ in trace metal chemistry; in particular, melanosomes from fossil vertebrate eyes are depleted in Zn and enriched in Cu relative to their extant counterparts. Whether these chemical differences are biological or taphonomic in origin is unknown, limiting our ability to use melanosome trace metal chemistry to test palaeobiological hypotheses. Here, we use maturation experiments on eye melanosomes from extant vertebrates and synchrotron rapid scan‐x‐ray fluorescence analysis to show that thermal maturation can dramatically alter melanosome trace element chemistry. In particular, maturation of melanosomes in Cu‐rich solutions results in significant depletion of Zn, probably due to low pH and competition effects with Cu. These results confirm fossil melanosome chemistry is susceptible to alteration due to variations in local chemical conditions during diagenesis. Maturation experiments can provide essential data on melanosome chemical taphonomy required for accurate interpretations of preserved chemical signatures in fossils.     

Oxidative stress in ARPE-19 cultures: Do melanosomes confer cytoprotection?

The pigment melanin has antioxidant properties that could theoretically reduce oxidative damage to the retinal pigment epithelium (RPE), perhaps protecting against retinal diseases with an oxidative stress component like age-related macular degeneration. To determine whether melanin confers cytoprotection on RPE cells, melanosomes or control particles were introduced by phagocytosis into the human cell line ARPE-19 and oxidative stress was induced chemically (H2O2 or tert-butyl hydroperoxide) or with visible light. Since the iron-binding capacity of melanin is important for its antioxidant function, experiments were performed to confirm that the melanosomes were not iron saturated. Cytotoxicity was assessed by measures of plasma or lysosomal membrane integrity, mitochondrial function, and cell-substrate reattachment. Oxidative stress protocols were critically evaluated to produce modest cytotoxicity, which might allow detection of a small cytoprotective effect as expected for melanosomes. Particle internalization alone had no effect on baseline metabolic activity or on major RPE antioxidants. Particles were tested in multiple oxidative stress experiments in which culture conditions known to affect stress-induced cytotoxicity, notably culture density, were varied. No testing condition or outcome measure revealed a consistent protective (or cytotoxic) effect of melanosomes, indicating that measures of lysosome stability or whole cell viability do not demonstrate an antioxidant role for RPE melanosomes. If the melanosome, an insoluble particle, performs a cytoprotective function within cells, its effects may be limited to the local environment of the organelle and undetectable by conventional methods.     

Loss of melanin by eye retinal pigment epithelium cells is associated with its oxidative destruction in melanolipofuscin granules

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.     

Pigment types in selected color genotypes of Asiatic sheep

The types and amounts of pigments in fibers from variously colored Tajik, Hissar, and Caracul sheep were determined by three methods: high-performance liquid chromatography, electron spin resonance spectroscopy, and light microscopic evaluation of melanosomes. In both dominant and recessive black lambs the color is due to eumelanin pigment. Brown and red phenotypes are the result of interaction of AWt and EBl, EBr, or EY alleles, and these colors are caused by mixtures of eumelanin and pheomelanin in varying ratios. The HPLC and ESR measurements detected these differences in melanin type, while direct characterization of melanosomes generally failed to distinguish between melanin type or relative ratio of melanin type.     

Fossilization of melanosomes via sulfurization

Fossil melanin granules (melanosomes) are an important resource for inferring the evolutionary history of colour and its functions in animals. The taphonomy of melanin and melanosomes, however, is incompletely understood. In particular, the chemical processes responsible for melanosome preservation have not been investigated. As a result, the origins of sulfur‐bearing compounds in fossil melanosomes are difficult to resolve. This has implications for interpretations of original colour in fossils based on potential sulfur‐rich phaeomelanosomes. Here we use pyrolysis gas chromatography mass spectrometry (Py‐GCMS), fourier transform infrared spectroscopy (FTIR) and time of flight secondary ion mass spectrometry (ToF‐SIMS) to assess the mode of preservation of fossil microstructures, confirmed as melanosomes based on the presence of melanin, preserved in frogs from the Late Miocene Libros biota (NE Spain). Our results reveal a high abundance of organosulfur compounds and non‐sulfurized fatty acid methyl esters in both the fossil tissues and host sediment; chemical signatures in the fossil tissues are inconsistent with preservation of phaeomelanin. Our results reflect preservation via the diagenetic incorporation of sulfur, i.e. sulfurization (natural vulcanization), and other polymerization processes. Organosulfur compounds and/or elevated concentrations of sulfur have been reported from melanosomes preserved in various invertebrate and vertebrate fossils and depositional settings, suggesting that preservation through sulfurization is likely to be widespread. Future studies of sulfur‐rich fossil melanosomes require that the geochemistry of the host sediment is tested for evidence of sulfurization in order to constrain interpretations of potential phaeomelanosomes and thus of original integumentary colour in fossils.