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.     

Melanosome diversity and convergence in the evolution of iridescent avian feathers—Implications for paleocolor reconstruction

Some of the most varied colors in the natural world are created by iridescent nanostructures in bird feathers, formed by layers of melanin‐containing melanosomes. The morphology of melanosomes in iridescent feathers is known to vary, but the extent of this diversity, and when it evolved, is unknown. We use scanning electron microscopy to quantify the diversity of melanosome morphology in iridescent feathers from 97 extant bird species, covering 11 orders. In addition, we assess melanosome morphology in two Eocene birds, which are the stem lineages of groups that respectively exhibit hollow and flat melanosomes today. We find that iridescent feathers contain the most varied melanosome morphologies of all types of bird coloration sampled to date. Using our extended dataset, we predict iridescence in an early Eocene trogon (cf. Primotrogon) but not in the early Eocene swift Scaniacypselus, and neither exhibit the derived melanosome morphologies seen in their modern relatives. Our findings confirm that iridescence is a labile trait that has evolved convergently in several lineages extending down to paravian theropods. The dataset provides a framework to detect iridescence with more confidence in fossil taxa based on melanosome morphology.     

Pigmented anatomy in Carboniferous cyclostomes and the evolution of the vertebrate eye

The success of vertebrates is linked to the evolution of a camera-style eye and sophisticated visual system. In the absence of useful data from fossils, scenarios for evolutionary assembly of the vertebrate eye have been based necessarily on evidence from development, molecular genetics and comparative anatomy in living vertebrates. Unfortunately, steps in the transition from a light-sensitive ‘eye spot’ in invertebrate chordates to an image-forming camera-style eye in jawed vertebrates are constrained only by hagfish and lampreys (cyclostomes), which are interpreted to reflect either an intermediate or degenerate condition. Here, we report—based on evidence of size, shape, preservation mode and localized occurrence—the presence of melanosomes (pigment-bearing organelles) in fossil cyclostome eyes. Time of flight secondary ion mass spectrometry analyses reveal secondary ions with a relative intensity characteristic of melanin as revealed through principal components analyses. Our data support the hypotheses that extant hagfish eyes are degenerate, not rudimentary, that cyclostomes are monophyletic, and that the ancestral vertebrate had a functional visual system. We also demonstrate integument pigmentation in fossil lampreys, opening up the exciting possibility of investigating colour patterning in Palaeozoic vertebrates. The examples we report add to the record of melanosome preservation in Carboniferous fossils and attest to surprising durability of melanosomes and biomolecular melanin.     

Support and challenges to the melanosomal casing model based on nanoscale distribution of metals within iris melanosomes detected by X‐ray fluorescence analysis

Melanin within melanosomes exists as eumelanin or pheomelanin. Distributions of these melanins have been studied extensively within tissues, but less often within individual melanosomes. Here, we apply X‐ray fluorescence analysis with synchrotron radiation to survey the nanoscale distribution of metals within purified melanosomes of mice. The study allows a discovery‐based characterization of melanosomal metals, and, because Cu is specifically associated with eumelanin, a hypothesis‐based test of the ‘casing model’ predicting that melanosomes contain a pheomelanin core surrounded by a eumelanin shell. Analysis of Cu, Ca, and Zn shows variable concentrations and distributions, with Ca/Zn highly correlated, and at least three discrete patterns for the distribution of Cu vs. Ca/Zn in different melanosomes – including one with a Cu‐rich shell surrounding a Ca/Zn‐rich core. Thus, the results support predictions of the casing model, but also suggest that in at least some tissues and genetic contexts, other arrangements of melanin may co‐exist.     

Evolutionary shifts in the melanin‐based color system of birds

Melanin pigments contained in organelles (melanosomes) impart earthy colors to feathers. Such melanin‐based colors are distributed across birds and thought to be the ancestral color‐producing mechanism in birds. However, we have had limited data on melanin‐based color and melanosome diversity in Palaeognathae, which includes the flighted tinamous and large‐bodied, flightless ratites and is the sister taxon to all other extant birds. Here, we use scanning electron microscopy and spectrophotometry to assess melanosome morphology and quantify reflected color for 19 species within this clade. We find that brown colors in ratites are uniquely associated with elongated melanosomes nearly identical in shape to those associated with black colors. Melanosome and color diversity in large‐bodied ratites is limited relative to other birds (including flightless penguins) and smaller bodied basal maniraptoran dinosaur outgroups of Aves, whereas tinamous show a wider range of melanosome forms similar to neognaths. The repeated occurrence of novel melanosome forms in the nonmonophyletic ratites suggests that melanin‐based color tracks changes in body size, physiology, or other life history traits associated with flight loss, but not feather morphology. We further anticipate these findings will be useful for future color reconstructions in extinct species, as variation in melanosome shape may potentially be linked to a more nuanced palette of melanin‐based colors.     

Melanin as a Target for Melanoma Chemotherapy: Pro‐oxidant Effect of Oxygen and Metals on Melanoma Viability

Melanoma cells have a poor ability to mediate oxidative stress, which may be attributed to constitutive abnormalities in their melanosomes. We hypothesize that disorganization of the melanosomes will allow chemical targeting of the melanin within. Chemical studies show that under oxidative conditions, synthetic melanins demonstrate increased metal affinity and a susceptibility to redox cycling with oxygen to form reactive oxygen species. The electron paramagnetic resonance (EPR)‐active 5,5′‐dimethyl‐pyrollidine N‐oxide spin adduct was used to show that binding of divalent Zn or Cu to melanin induces a pro‐oxidant response under oxygen, generating superoxide and hydroxyl radicals. A similar pro‐oxidant behaviour is seen in melanoma cell lines under external peroxide stress. Melanoma cultures grown under 95% O₂/5% CO₂ atmospheres show markedly reduced viability as compared with normal melanocytes. Cu‐ and Zn‐dithiocarbamate complexes, which induce passive uptake of the metal ions into cells, show significant antimelanoma activity. The antimelanoma effect of metal‐ and oxygen‐induced stress appears additive rather than synergistic; both treatments are shown to be significantly less toxic to melanocytes.     

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.     

Manassantin B inhibits melanosome transport in melanocytes by disrupting the melanophilin–myosin Va interaction

Human skin hyperpigmentation disorders occur when the synthesis and/or distribution of melanin increases. The distribution of melanin in the skin is achieved by melanosome transport and transfer. The transport of melanosomes, the organelles where melanin is made, in a melanocyte precedes the transfer of the melanosomes to a keratinocyte. Therefore, hyperpigmentation can be regulated by decreasing melanosome transport. In this study, we found that an extract of Saururus chinensis Baill (ESCB) and one of its components, manassantin B, inhibited melanosome transport in Melan‐a melanocytes and normal human melanocytes (NHMs). Manassantin B disturbed melanosome transport by disrupting the interaction between melanophilin and myosin Va. Manassantin B is neither a direct nor an indirect inhibitor of tyrosinase. The total melanin content was not reduced when melanosome transport was inhibited in a Melan‐a melanocyte monoculture by manassantin B. Manassantin B decreased melanin content only when Melan‐a melanocytes were co‐cultured with SP‐1 keratinocytes or stimulated by α‐MSH. Therefore, we propose that specific inhibitors of melanosome transport, such as manassantin B, are potential candidate or lead compounds for the development of agents to treat undesirable hyperpigmentation of the skin.     

Taphonomic experiments resolve controls on the preservation of melanosomes and keratinous tissues in feathers

Fossils are a key source of data on the evolution of feather structure and function through deep time, but their ability to resolve macroevolutionary questions is compromised by an incomplete understanding of their taphonomy. Critically, the relative preservation potential of two key feather components, melanosomes and keratinous tissue, is not fully resolved. Recent studies suggesting that melanosomes are preferentially preserved conflict with observations that melanosomes preserve in fossil feathers as external moulds in an organic matrix. To date, there is no model to explain the latter mode of melanosome preservation. We addressed these issues by degrading feathers in systematic taphonomic experiments incorporating decay, maturation and oxidation in isolation and combination. Our results reveal that the production of mouldic melanosomes requires interactions with an oxidant and is most likely to occur prior to substantial maturation. This constrains the taphonomic conditions under which melanosomes are likely to be fossilized. Critically, our experiments also confirm that keratinous feather structures have a higher preservation potential than melanosomes under a range of diagenetic conditions, supporting hitherto controversial hypotheses that fossil feathers can retain degraded keratinous structures.     

Assembly,Target‐Signaling and Intracellular Transport of Tyrosinase Gene Family Proteins in the Initial Stage of Melanosome Biogenesis

Assembly, target‐signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein‐3 (AP‐3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di‐leucine motif in their cytoplasmic tail, to which AP‐3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

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.     

The existence of apotyrosinase in the cytosol of Harding-Passey mouse melanoma melanocytes and characteristics of enzyme reconstitution by Cu(II)

This paper reports the effect of Cu(II) supplementation on the tyrosinase isozymes from Harding-Passey mouse melanoma. The dopa-oxidase activity of the microsomal and soluble isozymes is increased by incubation with Cu(II), whereas the activity of the unique 'in vivo' melanin-forming isozyme, bound to melanosomes, is not. Other divalent cations are ineffective in increasing the dopa-oxidase activity of tyrosinases. These results indicate the existence of a mixture of tyrosinase and apotyrosinase in the cytosol of melanocytes before reaching the melanosome. The paucity of Cu(II) in the cytosol could be one of the mechanisms of regulation contributing to avoid the formation of melanin outside the melanosome. Some kinetic characteristics of the enzymatic reconstitution of soluble and microsomal isozymes by Cu(II) are also studied, and the results suggest that the glycosylation of apotyrosinase during its maturation yields a conformational change favouring the binding of Cu(II) at the enzyme active site, by lowering the activation energy of the reconstitution reaction.     

Essential role of the molecular chaperone gp96 in regulating melanogenesis

Through a process known as melanogenesis, melanocyte produces melanin in specialized organelles termed melanosomes, which regulates pigmentation of the skin, eyes, and hair. Gp96 is a constitutively expressed heat shock protein in the endoplasmic reticulum whose expression is further upregulated upon ultraviolet irradiation. However, the roles and mechanisms of this chaperone in pigmentation biology are unknown. In this study, we found that knockdown of gp96 by RNA interference significantly perturbed melanin synthesis and blocked late melanosome maturation. Gp96 knockdown did not impair the expression of tyrosinase, an essential enzyme in melanin synthesis, but compromised its catalytic activity and melanosome translocation. Further, mice with melanocyte‐specific deletion of gp96 displayed decreased pigmentation. A mechanistic study revealed that the defect in melanogenesis can be rescued by activation of the canonical Wnt pathway, consistent with the critical roles of gp96 in chaperoning Wnt‐coreceptor LRP6. Thus, this work uncovered the essential role of gp96 in regulating melanogenesis.     

Melanosome degradation: fact or fiction

Our mini review summarizes what is known about the (bio)degradation of melanosomes. Unlike melanosome biogenesis where our knowledge enables us to explain it in molecular terms posing many interesting questions on the relation between lysosomes and melanosomes, melanosome degradation has remained 'terra incognita'. Observations at optical and ultrastructural levels describe the disintegration of melanosomes in the lysosomal compartment (in auto- and heterophagosomes). Histochemical studies suggest the participation of acid hydrolases in the process of melanosome degradation. Biochemical data confirm the ability of lysosomal hydrolases to degrade melanosome constituents except the melanin moiety. The similarity of melanin structure to that of polycyclic aromatic hydrocarbons suggests that melanin should be sensitive mainly, if not exclusively, to oxidative breakdown. In vitro melanin can indeed be decomposed by an oxidative attack and the degradation is accompanied by fluorescence and decreasing absorbance. From enzymes engaged in the biotransformation of polycyclic hydrocarbons only phagosomal NADPH oxidase meets the criteria (particularly as for compartmental and catalytic properties) to be involved in melanin biodegradation. The in vivo biodegradation of melanin has so far been clearly demonstrated in Aspergillus and fungi melanins.     

Pyridinyl imidazole compounds interfere with melanosomes sorting through the inhibition of Cyclin G-associated Kinase,a regulator of cathepsins maturation

Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. Pharmacological modulation of melanosomal transfer has recently gained much attention as a strategy for modifying normal or abnormal pigmentation. In this study, while investigating the impact of pyridinyl imidazole (PI) compounds, a class of p38 MAPK inhibitors, on melanocyte differentiation we observed that some, but not all PIs interfere with the physiological melanosome sorting producing a strong retention of melanin in the intracellular compartment associated with a general reduction of melanin synthesis. Electron microscopy studies illustrated an accumulation of melanosomes inside melanocytes with enrichment in immature melanosome at stages II and III at the end of dendrites. We identified cyclin G-associated kinase GAK, a protein expressed ubiquitously in various tissues, as the off-target responsible of intracellular melanin accumulation and we report evidence that reduced GAK-dependent cathepsin maturation is implicated in melanosome sorting deficiency. The co-regulation of GAK and cathepsin B and L expression with the melanogenic biosynthetic pathway in normal human melanocytes as well as in B16-F0 melanoma cells strengthen the idea that these proteins represent new possible targets for prevention and treatment of irregular pigmentation.     

Keratinocyte–Melanocyte Interactions During Melanosome Transfer

The epidermal–melanin unit is composed of one melanocyte and approximately 36 neighboring keratinocytes, working in synchrony to produce and distribute melanin. Melanin is synthesized in melanosomes, transferred to the dendrite tips, and translocated into keratinocytes, forming caps over the keratinocyte nuclei. The molecular and cellular mechanisms involved in melanosome transfer and the keratinocyte–melanocyte interactions required for this process are not yet completely understood. Suggested mechanisms of melanosome transfer include melanosome release and endocytosis, direct inoculation (‘injection’), keratinocyte–melanocyte membrane fusion, and phagocytosis. Studies of the keratinocyte receptor protease‐activated receptor‐2 (PAR‐2) support the phagocytosis theory. PAR‐2 controls melanosome ingestion and phagocytosis by keratinocytes and exerts a regulatory role in skin pigmentation. Modulation of PAR‐2 activity can enhance or decrease melanosome transfer and affects pigmentation only when there is keratinocyte–melanocyte contact. Moreover, PAR‐2 is induced by UV irradiation and inhibition of PAR‐2 activation results in the prevention of UVB‐induced tanning. The role of PAR‐2 in mediating UV‐induced responses remains to be elucidated.