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Loaches, Misgurnus anguillicaudatus, from Poyang Lake are a small economic fish, falling into three categories with typical blackspot characteristics, namely, big blackspot loaches (BBL), small blackspot loaches (SBL), and non-blackspot loaches (NBL), respectively. Pigment cell composition, pigment content, tyrosinase content and activity of three kinds of loaches were investigated. Our results showed that there were three types of skin pigment cells, namely, melanophores, xanthophores and iridophores. Melanophores in dorsum were more than in abdomen. Melanophore cytosomes in BBL were larger than those in SBL and NBL, and melanosomes were the largest in stage four. The melanophores in dorsal skin of SBL or NBL were small cell bodies, spindle-like, and in chain distribution. There was an extremely significant difference in melanin content in BBL between the dorsum and abdomen (P 0.05). In dorsal skin, tyrosinase content was the highest in BBL, and it was significantly lower in NBL than in BBL and SBL (P

Kaempferol, a representative flavonoid constituent of Sanguisorba officinalis, promotes melanogenesis, but the underlying mechanisms remain unknown. Here, we evaluated the effects of kaempferol on melanocytes morphology and behavior and determined the mechanisms regulating kaempferol-induced pigmentation. We observed that kaempferol increased melanin contents and dendritic length and stimulated melanocyte migration both in vitro and vivo. It significantly enhanced the expression of microphthalmia-associated transcription factor (MITF) and downstream enzymes of melanin biosynthesis—tyrosinase (TYR), tyrosinase-related protein (TRP-1), and dopachrome tautomerase (DCT). It also induced melanosome maturation (increased stage III and IV melanosomes) and melanin transfer to dendritic tips; this was evidenced as follows: kaempferol-treated melanocytes exhibited the perimembranous accumulation of HMB45-positive melanosomes and increased the expression of Rab27A, RhoA, and Cdc42, which improved melanosome transport to perimembranous actin filaments. These results jointly indicated that kaempferol promotes melanogenesis and melanocyte growth. Additionally, kaempferol stimulated the phosphorylation of P38/ERK MAPK and downregulated p-PI3K, p-AKT, and p-P70s6K expression. Pre-incubation with P38 (SB203580) and ERK (PD98059) signaling inhibitors reversed the melanogenic and dendritic effects and MITF expression. PI3K/AKT inhibitor augmented kaempferol-induced melanin content and dendrite length. In summary, kaempferol regulated melanocytes’ dendritic growth and melanosome quantity, maturation, and transport via P38/ERK MAPK and PI3K/AKT signaling pathways.

Melanogenesis is regulated by melanocytes, which synthesize the pigment melanin inside melanosomes; these melanosomes are exported through dendritic extensions to adjacent keratinocytes and result in skin coloration. Chemically modified tetracyclines (CMTs) are nonantimicrobial tetracyclines that retain the capacity to inhibit matrix metalloproteinases (MMPs) and have shown several biological benefits; in particular, CMT-3 [(4-dedimethylamino sancycline (SAN)] has emerged as a candidate for therapeutic benefits in our previous studies. However, to date, studies of the effects of CMT-3 or SAN on melanogenesis are lacking. We have previously reported the anti-melanogenic activity of CMT-308 (the 9-amino derivative of CMT-3). Herein, we have compared the three tetracycline analogs, doxycycline (DOX), SAN, and CMT-3, for their effects on melanogenesis using B16F10 mouse melanoma cells and have validated results in primary human melanocytes (HEMn-DP). DOX did not show any significant effects on intracellular melanin or melanosome export in DP cells while SAN was cytotoxic at high doses but without effects on melanogenesis at lower doses. However, CMT-3 showed a robust suppression of dendricity parameters (dendrite number, dendrite length, and proportion of dendritic cells) in DP cells which was associated, at least in part, with a significant reduction of intracellular tyrosinase activity. In spite of its inhibition of tyrosinase activity, CMT-3 had no significant effects on intracellular melanin levels, suggesting that it selectively targets melanosome export. Our results demonstrate a unique structure-activity relationship (SAR) for the effects of these compounds on melanogenesis and support the conclusion that removal of the 4-dimethylamino moiety confers the selective capacity to suppress melanosome export. Collectively, these results indicate that CMT-3 might be a candidate for diminishing hyperpigmentation skin disorders.

Extremely limited drug retention and depigmentation represent the greatest barriers against vitiligo treatment advancement. Here, inspired by biological melanosomes, the primary melanin transporter, we developed biomimetic melanosomes to combat reactive oxygen species (ROS)-mediated melanocyte damage and depigmentation. Briefly, methylprednisolone (MPS) and melanin-mimicking polydopamine (PDA) were encapsulated inside lysine-proline-valine (KPV)-modified deformable liposomes (KPV-Lipos). Owing to their phospholipid bilayer flexibility and the specific affinity for melanocortin 1 receptor (MC1R), KPV-Lipos exhibited 1.43-fold greater skin deposition than traditional liposomes. The binding of KPV and its receptor also contributed to activating the cAMP-tyrosinase (TYR) signaling pathway, improving the endogenous melanin content. In addition, PDA mimicked melanosomes as it effectively increased the exogenous melanin content and scavenged ROS. Meanwhile, MPS inhibited inflammatory cytokine secretion, limiting the depigmented area. Ultimately, the biomimetic melanosomes affected the skin color of mice with H2O2-induced vitiligo. These melanosomes show potential as a universal platform for the self-supply of melanin by self-driven melanin synthesis with exogenous supplementation. Furthermore, this study offers ideas for the production of artificial packed melanosome substitutes for melanocyte-related diseases.

Human skin is a highly efficient self-renewing barrier that is critical to withstanding environmental insults. Undifferentiated keratinocyte stem cells reside in the basal layer of the epidermis and in hair follicles that continuously give rise to progenies ensuring epidermal turnover and renewal. Ultraviolet (UV) radiation is a proven cause of skin keratinocyte cancers, which preferentially occur at sun-exposed areas of the skin. Fortunately, melanocytes produce melanin that is packaged in specific organelles (termed melanosomes) that are then delivered to nearby keratinocytes, endowing the recipient cells with photoprotection. It has long been thought that melanosome transfer takes place stochastically from melanocytes to keratinocytes via an as-yet-unrecognized manner. However, recent studies have indicated that melanosomes are distributed regionally in the basal layer of the skin, affording localized intensive photoprotection for progenitor keratinocytes and stem cells that reside in the microenvironment of the basal epidermis. In this review, we summarize current knowledge about molecular and cellular mechanisms that are responsible for the selective transfer and exclusive degradation of melanosomes in the epidermis, emphasizing implications for skin carcinogenesis.

In a previous study, we observed that the hair color of Japanese females darkens with age and that the causes of this are the increase in melanosome size, the amount of melanin, and the mol% of 5,6-dihydroxyindole (DHI) which has a high absorbance. In this study, we extended the same analyses to male hair to examine the sex differences in hair color, melanin composition, and melanosome morphology. Male hair also tended to darken with age, but it was darker than female hair in those of younger ages. Although there was no age dependence of DHI mol% in male hair, as with female hair, the melanosomes’ sizes enlarged with age, the total melanin amount increased, and these findings were correlated with hair color. The analyses, considering age dependence, revealed that there were significant sex differences in the ratio of absorbance of dissolved melanin at the wavelength of 650 nm to 500 nm, in pheomelanin mol%, and in melanosome morphology parameters such as the minor axis. This may be the cause of the sex differences in hair color. Furthermore, the factors related to hair color were analyzed using all the data of the male and female hairs. The results suggested that total melanin amount, pheomelanin mol%, and DHI mol% correlated with hair color.

Skin whitening has become one of the aesthetic problems among humans around the world, especially on women which is highly attention to skin beauty and whitening. Many of the plant extracts in oral energy drinks have whitening effects and good taste, which can support the health of the skin. We use the National Center for Biotechnology Information (NCBI) as a search engine to find literatures about skin whitening, oral plant extracts. This review mainly introduces the mechanism of skin whitening and the application of oral plant extracts in skin whitening. Skin whitening at melanin synthesis, melanosome transport, metabolism, oxidative stress, UV stimulation, other environmental stimulation inseparable. Many oral plant extracts are natural tyrosine inhibitors. The review concludes the melanin synthesis and transporting, pathway of melanin synthesis, whitening mechanism and the function of whitening on Green tea polyphenols, Rose Petal Extract (Rosa gallica), Pears, Peony, Rosa roxburghii Tratt, Olive (Olea europaea L.), Pomegranates, Phyllanthus emblica L., Mulberry extract, Açaí berry, saussurea involucrate flavonoids extract, etc. These will provide a strong foundation for the application of oral plant extracts in skin whitening. Furthermore, the mixture with function of skin whitening/ skin anti-aging / anti-inflammatory antioxidant oral plant extract can be a good candidate for further development on skin care product.

There is an increasing recognition of ethnic dermatology to reflect the increase in skin of colour (SOC) populations in the UK. Hyperpigmentary disorder is one of the commonest skin concerns in SOC but there has been limited training available in this field of dermatology. Variations in skin colour are genetically determined by the amount of melanin content, the eumelanin/pheomelanin ratio and the size of melanosomes, but is also influenced by other factors such as hormones and extrinsic factors such as ultraviolet radiation. Hyperpigmentation is a broad term to describe increased pigmentation in the skin, and making a correct diagnosis is an important first step in the successful management of hyperpigmentary disorders. A systematic approach based on the disease pathogenesis (e.g. reactive vs. nonreactive, increased melanin vs. increased number of cells or epidermal vs. dermal pigmentation) aided by a detailed history and clinical examination is the best way to diagnose a hyperpigmentary disorder. Based on its pathogenesis, management can be planned. For epidermal hyperpigmentation caused by increased melanin, topical skin-lightening agents targeting inhibition of tyrosinase or melanosome transfer and promotion of keratinocyte turnover can be used. Hydroquinone-containing cream is the gold-standard treatment for epidermal hyperpigmentation. Alternative treatments include laser toning or chemical peels. However, increased dermal pigmentation is more challenging to target with topical treatments. If hyperpigmentation is due to increased numbers of melanocytes or keratinocytes, high-fluence laser is the most appropriate treatment method.

Color is a phenotypic trait of utmost importance, particularly in birds, which are known for their diverse color signals and color-producing mechanisms including pigment-based colors, light scattering from nanostructured feather tissues and combinations thereof. Bright iridescent plumage colors of hummingbirds are caused by light scattering by an organized array of flattened, pigment organelles, containing air-filled vesicles, called melanosomes. These hollow platelets are organized in multilayer arrays that contain numerous sharp air/melanin refractive index interfaces, producing brilliant iridescent colors. Despite their ecological significance and potential for inspiration of new optical materials, how platelets form and spatially arrange in nanostructures in growing feathers remains unknown. Here, we tested the hypothesis that melanosome formation and organization occurs mostly through passive self-assembly processes by assembling a developmental time series of growing hummingbird feathers using optical and electron microscopy. We show that hummingbird platelets contain air bubbles or vesicles upon their formation in pigment-producing cells, melanocytes. When melanosomes are transferred to neighboring keratinocytes (the cells shaping barbule structure) they drastically expand in size; and variation in this enlargement appears to be driven by physical constraints caused by the placement of the melanosomes within the barbule plate and their proximity to other melanosomes. As the barbule elongates and narrows, polymerizing feather corneous beta-protein orients melanosomes unilaterally, forcing them into a stacked configuration. These results reveal potentially novel forces driving the self-assembly of the nanostructures producing some of the brightest colors in nature.

Skin pigmentation depends not only on melanosomes produced by melanocytes but also on the migration of melanosomes to adjacent keratinocytes and their distribution on the skin surface. The inhibitory effects of cardanol diene (CADI), a major phenolic compound extracted from cashew nut shell liquid (CNSL), on melanin transfer in B16F10-HaCAT co-culture cells and a crude extracted compound of cashew nut shell oil (CNCE) on melanin production in zebrafish were investigated. β-arbutin was used as a positive control. CADI inhibited the expression levels of the melanin phagocytosis-associated protein PAR-2 and filopodia-associated protein NMDAR1, and the effect was better than that of β-arbutin at the same concentration. To further study the inhibitory mechanism of CADI in melanin transfer, fluorescence microspheres were used to simulate melanin and co-culture with keratinocytes. The results indicated that CADI inhibited melanin transfer through suppressing keratinocyte phagocytosis, dendrite formation and filopodia three channels. We also found that CNCE inhibited melanin production in zebrafish and showed significant inhibitory effects on TYR, DCT and α-MSH. Thus, the natural components extracted from CNSL effectively inhibited skin pigmentation, providing insight into the application of new anti-melanin drugs in cosmetics and medicine.

SynopsisMelanins, the main pigments of the skin and hair in mammals, are synthesized within membrane-bound organelles of melanocytes called melanosomes. Melanosome structure and function are determined by a cohort of resident transmembrane proteins, many of which are expressed only in pigment cells and localize specifically to melanosomes. Defects in the genes that encode melanosome-specific proteins or components of the machinery required for their transport in and out of melanosomes underlie various forms of ocular or oculocutaneous albinism, characterized by hypopigmentation of the hair, skin, and eyes and by visual impairment. We review major components of melanosomes, including the enzymes that catalyze steps in melanin synthesis from tyrosine precursors, solute transporters that allow these enzymes to function, and structural proteins that underlie melanosome shape and melanin deposition. We then review the molecular mechanisms by which these components are biosynthetically delivered to newly forming melanosomes—many of which are shared by other cell types that generate cell type-specific lysosome-related organelles. We also highlight unanswered questions that need to be addressed by future investigation.

Infantile nystagmus syndrome (INS) denominates early-onset, involuntary oscillatory eye movements with different etiologies. Nystagmus is also one of the symptoms in oculocutaneus albinism (OCA), a heterogeneous disease mainly caused by defects in melanin synthesis or melanosome biogenesis. Dopachrome tautomerase (DCT, also called TYRP2) together with tyrosinase (TYR) and tyrosin-related protein 1 (TYRP1) is one of the key enzymes in melanin synthesis. Although DCT´s role in pigmentation has been proven in different species, until now only mutations in TYR and TYRP1 have been found in patients with OCA. Detailed ophthalmological and orthoptic investigations identified a consanguineous family with two individuals with isolated infantile nystagmus and one family member with subtle signs of albinism. By whole-exome sequencing and segregation analysis, we identified the missense mutation c.176G > T (p.Gly59Val) in DCT in a homozygous state in all three affected family members. We show that this mutation results in incomplete protein maturation and targeting in vitro compatible with a partial or total loss of function. Subsequent screening of a cohort of patients with OCA (n = 85) and INS (n = 25) revealed two heterozygous truncating mutations, namely c.876C > A (p.Tyr292*) and c.1407G > A (p.Trp469*), in an independent patient with OCA. Taken together, our data suggest that mutations in DCT can cause a phenotypic spectrum ranging from isolated infantile nystagmus to oculocutaneous albinism.

Human skin and hair colour are due to a pigment, melanin, which is produced at the dermal epidermal level by specialized cells, the melanocytes, in a process named melanogenesis.The melanocytes are derived from their precursors, melanoblasts, with migrate to their definitive locations in the course of the final steps of embryogenesis. The melanocytes produce melanosomes, small organelles where melanin synthesis will take place. There are four stages of development of melanosomes, and only from the third stage onward melanosomes can synthesize melanin. Then, the transfer of melanin from the melanocytes to the keratinocytes can take place. According to the individuals, there is a phenotypic diversity of pigmentation, which is described in Fitzpatrick classification, and due to the ratio eumelanin (dark melanin) and phaeomelanin (reddish melanin) produced by the melanocytes. Melanogenesis is quite a complex series of chemical reactions, along which three enzymes, tyrosinase (TYR), tyrosinase-related protein 1 (TRP1) and DOPAchrome tautomerase (DCT) are absolutely required. Other enzymes also play a role in this process.Melanocortin-1 receptor (MC1R) and Microphthalmia-associated transcription factor (MITF) also play a pivotal role in the stimulation (or not) of the melanocytes. Skin pigmentation is regulated by a series of intrinsic factors such as peptides, cytokines, prostaglandins, NO, but also oestrogens. On the other hand, UV radiation (UVR) is a potent extrinsic regulator of melanogenesis. In this paper, it was emphasized on the importance of the paracrine regulation of skin pigmentation. The major role of keratinocytes is well known, but other skin cells like fibroblast, immune cells or endothelial cells are of major importance in this cell-to-cell communication with the melanocytes and will regulate melanogenesis.Finally, a brief glossary was proposed about various existing melanins.

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on β-catenin activity, revealing a function of the Wnt/β-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer., Aktary et al. identify novel roles for the dynein light chain Dynlt3 in melanosome transport, maturation, and transfer to keratinocytes. They also find that the Wnt/βcatenin signalling pathway controls Dynlt3 levels and thus also contributes to the regulation of melanocyte transport and skin pigmentation.

Background and objectives In this study, the threshold fluences for disrupting the melanosomes for pigmented skin lesion treatment were determined using a 755-nm picosecond laser for clinical use. Based on the melanosome disruption thresholds, incident fluences corresponding to the target lesion depths were evaluated in silico for different laser spot sizes. Study design/materials and methods Melanosome samples were isolated from porcine eyes as alternative samples for human cutaneous melanosomes. The isolated melanosomes were exposed to 755-nm picosecond laser pulses to measure the mean particle sizes by dynamic light scattering and confirm their disruption by scanning electron microscopy. The threshold fluences were statistically determined from the relationships between the irradiated fluences and the mean particle sizes. Incident fluences of picosecond laser pulses for the disruption of melanosomes located at different depths in skin tissue were calculated through a light transport simulation using the obtained thresholds. Results The threshold fluences of 550- and 750-picosecond laser pulses were determined to be 2.19 and 2.49 J/cm2 , respectively. The numerical simulation indicated that appropriate incident fluences of picosecond laser pulses differ depending on the depth distribution of the melanosomes in the skin tissue, and large spot sizes are desirable for disrupting the melanosomes more deeply located within the skin tissue. Conclusion The threshold fluences of picosecond laser pulses for melanosome disruption were determined. The incident fluence analysis based on the thresholds for melanosome disruption provides valuable information for the selection of irradiation endpoints for picosecond laser treatment of pigmented skin lesions. Lasers Surg. Med. © 2021 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Cutaneous melanoma tumors are heterogeneous and show diverse responses to treatment. Identification of robust molecular biomarkers for classifying melanoma tumors into clinically distinct and homogenous subtypes is crucial for improving the diagnosis and treatment of the disease. In this study, we present a classification of melanoma tumors into four subtypes with different survival profiles based on three distinct gene expression signatures: keratin, immune, and melanogenesis. The melanogenesis expression pattern includes several genes that are characteristic of the melanosome organelle and correlates with worse survival, suggesting the involvement of melanosomes in melanoma aggression. We experimentally validated the secretion of melanosomes into surrounding tissues by melanoma tumors, which potentially affects the lethality of metastasis. We propose a simple molecular decision tree classifier for predicting a tumor’s subtype based on representative genes from the three identified signatures. Key predictor genes were experimentally validated on melanoma samples taken from patients with varying survival outcomes. Our three-pattern approach for classifying melanoma tumors can contribute to advancing the understanding of melanoma variability and promote accurate diagnosis, prognostication, and treatment.

Tyrosinase catalyzes the rate-limiting step in melanin synthesis. Melanin is synthesized from l-tyrosin in the melanosomes, where tyrosinase and other melanogenic factors are recruited via the vesicle transport system. Genetic and biochemical approaches have revealed a correlation between impairments in the vesicle transport system and albinism. However, the specificity of the individual transport systems for the corresponding melanogenic factors has not been well elucidated yet. Here, we report that the thioxothiazolidin derivative, 4-OST (4-[(5E)-5-[(4-fluorophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]-4-azatricyclo [5.2.1.02 ,6]dec-8-ene-3,5-dione: CAS RN. 477766-87-3) strongly inhibited melanogenesis in mouse melanoma B16F10 cells. 4-OST reduces tyrosinase protein levels without affecting its messenger RNA levels or enzymatic activity. Although a reduction in tyrosinase protein level was observed in the presence of a protein synthesis inhibitor, the reduction may be coupled with protein synthesis. Similarly, GIF-2202 (a derivative of 4-OST) lowers tyrosinase protein levels without affecting the levels of another melanogenic enzyme, tyrosinase-related protein 1 (TYRP1) level. The reduction in tyrosinase protein level is associated with an increase in the levels of the lysosomal proteinase cathepsin S. Chloroquine, a lysosome inhibitor, restored the tyrosinase protein level downregulated by GIF-2202, although no effects of other inhibitors (against proteasome, autophagy, or exocytosis) were observed. In addition, GIF-2202 segregated the immunofluorescence signals of tyrosinase from those of TYRP1. Chloroquine treatment resulted in co-localization of tyrosinase and cathepsin S signals near the perinuclear region, suggesting that 4-OST and GIF-2202 may alter the destination of the tyrosinase vesicle from the melanosome to the lysosome. 4-OST and GIF-2202 can be new tools for studying the tyrosinase-specific vesicle transport system.

To investigate the ultrastructural and clinical characteristics of melanoma of the central nervous system (CNS). The clinical and electron microscopy pathology data of nine patients with melanoma surveyed from 1993 to 2017 were analyzed. All the CNS melanomas were confirmed by transmission electron microscopy (TEM), including eight cases of primary melanomas and one case of metastatic melanoma. In this study, four stage II melanosomes were intracranial space-occupying, three of which were malignant melanoma, the other one was melanoma. Among the five stage IV melanosomes, four cases were intraspinal space-occupying, the other one was intracranial space-occupying, and the pathological diagnoses were all melanoma. At present, TEM is an important tool for the diagnosis of CNS melanomas. Malignant melanoma has high malignancy and recurrence rate and poor prognosis, while benign melanoma with relatively low recurrence rate, so we speculate that patients with mainly immature melanosomes are more likely to exhibit recurrence.

Purpose Numerous small hyperreflective dots (HRDs) can be seen within the hyporeflective layer between the ellipsoid zone (EZ) and the interdigitation zone (IZ) on C‐scan spectral‐domain optical coherence tomography (SD‐OCT) with a yet unknown variation under light conditions. The aim of this study was to explore light‐induced SD‐OCT changes in these HRDs. Methods The study subjects were randomly assigned to two groups: Group 1 experienced a dark adaptation protocol followed by intense retinal photobleaching, while Group 2, serving as the control group, was exposed to constant ambient light without any variation. The number of HRDs was automatically counted. Results Twenty healthy volunteers were prospectively included. The number of HRDs differed significantly over time (p = 0.0013). They decreased in Group 1 after dark adaptation and retinal photobleaching before returning to baseline levels 30 min later; conversely, they remained relatively constant in Group 2 throughout the study (p

Peptides are short chain of amino acids linked by peptide bonds. They are widely used as effective and biocompatible active ingredients in cosmetic industry. In this study, we developed novel peptide mixture and identified its anti-pigmentation effect on melanocytes and keratinocytes. Our results revealed that peptide mixture inhibited melanosome biogenesis through the regulation of microphthalmia-associated transcription factor, a key factor of melanogenesis in melanocytes. And we observed that peptide mixture inhibited melanosome uptake through the reduction of protease-activated receptor 2, a phagocytosis-related receptor in keratinocytes. Furthermore, peptide mixture activated autophagy system resulting in degradation of transferred melanosomes in keratinocytes. The anti-pigmentation effect of multi-targeting peptide mixture was assessed in a human skin equivalent model (MelanoDerm). Melanin contents in epidermal layer were significantly decreased by topical treatment of peptide mixture, suggesting that it can be applied as a novel cosmetics material having a whitening function.

Melanogenesis is a melanin-forming process responsible for protecting the skin against ultraviolet radiation damage. An excess production of melanin, however, may result in hyperpigmentation (darkening of the skin) to adverse dermatological effects (freckles, solar lentigines, and melasma) and skin cancer. These hyperpigmentary skin disorders may also have a major effect on a person's appearance and could even result in emotional and mental distress, as well as a diminished quality of life. A large number of melanogenesis inhibitors have been discovered, but most of them appeared to have undesirable side effects. Therefore, in order to better understand the mechanisms of hyperpigmentary skin disorders and to establish effective and safe melanogenesis inhibitors, more fundamental research is needed. Apart from tyrosinase blockers, there are also alternative approaches that involve the manipulation of melanogenesis regulatory pathway such as α-melanocyte-stimulating hormone blockers, melanosome transferase inhibitors, and cytokines. This review abridges data on the different melanogenesis inhibitors and depigmentation agents from both natural and synthetic agents from the last few years.

Dowling-Degos disease is a rare autosomal dominant genodermatosis. It is characterized by acquired reticulate hyperpigmentation over the flexures, comedone-like follicular papules, and pitted perioral scars that usually develop during adulthood. Mutations in genes affecting melanosome transfer, and melanocyte and keratinocyte differentiation have been implicated in the pathogenesis of this disease. These genes include KRT5, POFUT1, POGLUT1 and, most recently, PSENEN. Dowling-Degos disease can be found in isolation or with other associated findings, most notably hidradenitis suppurativa. This condition belongs to a spectrum of conditions that all result in reticulate hyperpigmentation that at times are hard to distinguish from each other. The most closely linked entity is Galli-Galli, which is clinically indistinguishable from Dowling-Degos disease and can only be distinguished by the presence of acantholysis on microscopy. Unfortunately, Dowling-Degos disease is generally progressive and recalcitrant to treatment.

Hyperpigmentation and hypopigmentation are two manifestations of skin pigmentation diseases. Recent studies have shown that autophagy is involved in the development of skin pigmentation diseases. The melanosome is a lysosome-related organelle characterized by the production of melanin. The autophagosome-lysosome degradation pathway exhibits a characteristic cell renewal function. The functions of melanosomes and autophagosomes intersect and the vesicle transport pathway mediates both autophagosome and melanosome formation, which may involve different regulatory protein complexes. Current studies have revealed that several autophagy-related regulators of autophagosome formation are involved in melanosome formation and maturation and also regulate melanogenesis, and that melanosomes can be degraded via autophagy in melanocytes. Autophagy is also involved in regulating the living environment of melanocytes. Understanding the effects of autophagy on pigmentation may support our understanding of pigmentation diseases. This article reviews the relationship between autophagy and pigmentation in melanocytes.

SLC45A2 encodes a putative transporter expressed primarily in pigment cells. SLC45A2 mutations cause oculocutaneous albinism type 4 (OCA4) and polymorphisms are associated with pigmentation variation, but the localization, function, and regulation of SLC45A2 and its variants remain unknown. We show that SLC45A2 localizes to a cohort of mature melanosomes that only partially overlaps with the cohort expressing the chloride channel OCA2. SLC45A2 expressed ectopically in HeLa cells localizes to lysosomes and raises lysosomal pH, suggesting that in melanocytes SLC45A2 expression, like OCA2 expression, results in the deacidification of maturing melanosomes to support melanin synthesis. Interestingly, OCA2 overexpression compensates for loss of SLC45A2 expression in pigmentation. Analyses of SLC45A2- and OCA2-deficient mouse melanocytes show that SLC45A2 likely functions later during melanosome maturation than OCA2. Moreover, the light skin-associated SLC45A2 allelic F374 variant restores only moderate pigmentation to SLC45A2-deficient melanocytes due to rapid proteasome-dependent degradation resulting in lower protein expression levels in melanosomes than the dark skin-associated allelic L374 variant. Our data suggest that SLC45A2 maintains melanosome neutralization that is initially orchestrated by transient OCA2 activity to support melanization at late stages of melanosome maturation, and that a common allelic variant imparts reduced activity due to protein instability.

Dozens of genes contribute to the vast variation in human pigmentation. Many of these encode proteins that localize to the melanosome, the lysosome-related organelle that synthesizes pigment, but have unclear functions 1,2. Here, we describe the MelanoIP method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use it to study MFSD12, a transmembrane protein of unknown molecular function that when suppressed causes darker pigmentation in mice and humans 3,4. We find that MFSD12 is required to maintain normal levels of cystine, the oxidized dimer of cysteine, in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation 5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes, and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal storage disease caused by inactivation of the lysosomal cystine exporter CTNS (Cystinosin)7–9. Thus, MFSD12 is an essential component of the long-sought cysteine importer for melanosomes and lysosomes.

Melanosomes are specialized intracellular organelles that produce and store melanin pigments in melanocytes, which are present in several mammalian tissues and organs, including the skin, hair, and eyes. Melanosomes form and mature stepwise (stages I-IV) in melanocytes and then are transported toward the plasma membrane along the cytoskeleton. They are subsequently transferred to neighboring keratinocytes by a largely unknown mechanism, and incorporated melanosomes are transported to the perinuclear region of the keratinocytes where they form melanin caps. Melanocytes also extend several dendrites that facilitate the efficient transfer of the melanosomes to the keratinocytes. Since the melanosome biogenesis, transport, and transfer steps require multiple membrane trafficking processes, Rab GTPases that are conserved key regulators of membrane traffic in all eukaryotes are crucial for skin and hair pigmentation. Dysfunctions of two Rab isoforms, Rab27A and Rab38, are known to cause a hypopigmentation phenotype in human type 2 Griscelli syndrome patients and in chocolate mice (related to Hermansky-Pudlak syndrome), respectively. In this review article, I review the literature on the functions of each Rab isoform and its upstream and downstream regulators in mammalian melanocytes and keratinocytes.

Significance Reptiles exhibit a spectacular diversity of skin colors generated by interactions among black melanophores, red and yellow xanthophores, as well as iridophores producing structural colors. Here, we use the corn snake to investigate the generative mechanisms of skin colors beyond the zebrafish model. We perform sequencing and annotation of a nearly chromosome-quality genome of the corn snake, followed by mapping-by-sequencing and identification of a mutation in the lysosomal trafficking regulator gene (LYST) in the lavender variant with strongly affected coloration. Further analyses indicate that color-producing organelles of all chromatophores are substantially impacted in the LYST mutant, indicating that not only melanosomes, but also xanthosomes and iridosomes, are all lysosome-related organelles., Reptiles exhibit a spectacular diversity of skin colors and patterns brought about by the interactions among three chromatophore types: black melanophores with melanin-packed melanosomes, red and yellow xanthophores with pteridine- and/or carotenoid-containing vesicles, and iridophores filled with light-reflecting platelets generating structural colors. Whereas the melanosome, the only color-producing endosome in mammals and birds, has been documented as a lysosome-related organelle, the maturation paths of xanthosomes and iridosomes are unknown. Here, we first use 10x Genomics linked-reads and optical mapping to assemble and annotate a nearly chromosome-quality genome of the corn snake Pantherophis guttatus. The assembly is 1.71 Gb long, with an N50 of 16.8 Mb and L50 of 24. Second, we perform mapping-by-sequencing analyses and identify a 3.9-Mb genomic interval where the lavender variant resides. The lavender color morph in corn snakes is characterized by gray, rather than red, blotches on a pink, instead of orange, background. Third, our sequencing analyses reveal a single nucleotide polymorphism introducing a premature stop codon in the lysosomal trafficking regulator gene (LYST) that shortens the corresponding protein by 603 amino acids and removes evolutionary-conserved domains. Fourth, we use light and transmission electron microscopy comparative analyses of wild type versus lavender corn snakes and show that the color-producing endosomes of all chromatophores are substantially affected in the LYST mutant. Our work provides evidence characterizing xanthosomes in xanthophores and iridosomes in iridophores as lysosome-related organelles.

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Tyrosinase is an important enzyme in controlling the formation of melanin in melanosome, and plays a key role in the pigmentation of hair and skin. The abnormal expression or activation of tyrosinase is associated with several diseases such as albinism, vitiligo, melanoma and Parkinson disease. Excessive deposition of melanin could cause diseases such as freckles and brown spots in the human body, and it is also closely related to browning of fruits and vegetables and insect molting. Detecting and inhibiting the activity of tyrosinase is of extraordinary value in the progress of diagnosis and treatment of these diseases. Therefore, many selective optical detection probes and small molecular inhibitors have been developed, and have made significant contributions to the basic and clinical research on these diseases. In this paper, the detection and inhibition of tyrosinase and their application in whitening products are reviewed, with special emphasis on development of fluorescent probes and inhibitors. Hopefully, this review will help design more efficient and sensitive tyrosinase probes and inhibitors, as well as shed light on novel treatment of diseases such as melanoma., Graphical abstract Image 1, Highlights • The abnormal expression or activation of tyrosinase is the pathogenesis of several diseases such as albinism, vitiligo, and melanoma. • Detecting and inhibiting tyrosinase activity is of great value in the diagnosis and treatment of these diseases. • The detection/inhibition of tyrosinase and its application in whitening products are reviewed, with special emphasis on probes/inhibitors.

Hair greying (canities) is one of the earliest, most visible ageing-associated phenomena, whose modulation by genetic, psychoemotional, oxidative, senescence-associated, metabolic and nutritional factors has long attracted skin biologists, dermatologists, and industry. Greying is of profound psychological and commercial relevance in increasingly ageing populations. In addition, the onset and perpetuation of defective melanin production in the human anagen hair follicle pigmentary unit (HFPU) provides a superb model for interrogating the molecular mechanisms of ageing in a complex human mini-organ, and greying-associated defects in bulge melanocyte stem cells (MSCs) represent an intriguing system of neural crest-derived stem cell senescence. Here, we emphasize that human greying invariably begins with the gradual decline in melanogenesis, including reduced tyrosinase activity, defective melanosome transfer and apoptosis of HFPU melanocytes, and is thus a primary event of the anagen hair bulb, not the bulge. Eventually, the bulge MSC pool becomes depleted as well, at which stage greying becomes largely irreversible. There is still no universally accepted model of human hair greying, and the extent of genetic contributions to greying remains unclear. However, oxidative damage likely is a crucial driver of greying via its disruption of HFPU melanocyte survival, MSC maintenance, and of the enzymatic apparatus of melanogenesis itself. While neuroendocrine factors [e.g. alpha melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), ß-endorphin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH)], and micropthalmia-associated transcription factor (MITF) are well-known regulators of human hair follicle melanocytes and melanogenesis, how exactly these and other factors [e.g. thyroid hormones, hepatocyte growth factor (HGF), P-cadherin, peripheral clock activity] modulate greying requires more detailed study. Other important open questions include how HFPU melanocytes age intrinsically, how psychoemotional stress impacts this process, and how current insights into the gerontobiology of the human HFPU can best be translated into retardation or reversal of greying.

Age‐related macular degeneration (AMD) is a multifactorial disease, which is characterized by loss of central vision, affecting one in three people by the age of 75. The Y402H polymorphism in the complement factor H (CFH) gene significantly increases the risk of AMD. We show that Y402H‐AMD‐patient‐specific retinal pigment epithelium (RPE) cells are characterized by a significant reduction in the number of melanosomes, an increased number of swollen lysosome‐like‐vesicles with fragile membranes, Cathepsin D leakage into drusen‐like deposits and reduced lysosomal function. The turnover of C3 is increased significantly in high‐risk RPE cells, resulting in higher internalization and deposition of the terminal complement complex C5b‐9 at the lysosomes. Inhibition of C3 processing via the compstatin analogue Cp40 reverses the disease phenotypes by relieving the lysosomes of their overburden and restoring their function. These findings suggest that modulation of the complement system represents a useful therapeutic approach for AMD patients associated with complement dysregulation., Schematic presentation of autophagy‐lysosomal pathway dysfunction in Y402H‐AMD RPE cells and the impact of Cp40 in restoring the lysosomal function.

Four cases of a rare melanotic variant of malignant nerve sheath tumour (MNST) in dogs are described. All four cases presented with neurological clinical signs due to multicentric, intradural, intra- and extraparenchymal neoplasms that surrounded the spinal and cranial nerves and infiltrated the adjacent spinal cord and brain. The dogs were young (3 months to 3 years of age), all were female and four different breeds were represented. Characteristic histological features were interweaving fascicles of spindle-shaped cells, sometimes with an architecture reminiscent of Antoni A and B patterns. Some spindle cells showed prominent cytoplasmic melanin pigmentation and such cells were positive by Masson-Fontana stain. Immunohistochemistry performed in three cases was positive for S100 and vimentin, strongly positive for melan A in the melanized cells and negative for glial fibrillary acidic protein and periaxin. Non-melanized cells did not express melan A. Transmission electron microscopy findings in one case were consistent with a peripheral nerve sheath tumour and demonstrated cytoplasmic pre-melanosomes and melanosomes. Melanotic variants of MNSTs are rare in animals with only a solitary report of two previous canine cases in the literature.

The human eye is made up of multiple layers of pigmented tissues that have in componence melanin. In the eye, one can separate melanosomes from various embryonic origins. The pigment-producing cells in the stroma of the iris, ciliary body and the choroid (uveal melanocytes) are neural crest derivatives. On the other hand, ciliary, iris and retinal pigment epithelial cells are developed from the neural ectoderm. One universally accepted role of melanin is to react as neutral-density filter in scattering light. Melanin acts as a free radical stabilizer and has the ability to absorb near-infrared, visible light and UV radiation. This paper reviews the current knowledge on ocular melanin, including ocular melanogenesis, roles of melanin in retinal metabolic processes and some imaging techniques that identify melanin in the retina.

Megakaryocyte-derived platelets and endothelial cells store their hemostatic cargo in α- and δ-granules and Weibel-Palade bodies, respectively. These storage granules belong to the lysosome-related organelles (LROs), a heterogeneous group of organelles that are rapidly released following agonist-induced triggering of intracellular signaling pathways. Following vascular injury, endothelial Weibel-Palade bodies release their content into the vascular lumen and promote the formation of long VWF (von Willebrand factor) strings that form an adhesive platform for platelets. Binding to VWF strings as well as exposed subendothelial collagen activates platelets resulting in the release of α- and δ-granules, which are crucial events in formation of a primary hemostatic plug. Biogenesis and secretion of these LROs are pivotal for the maintenance of proper hemostasis. Several bleeding disorders have been linked to abnormal generation of LROs in megakaryocytes and endothelial cells. Recent reviews have emphasized common pathways in the biogenesis and biological properties of LROs, focusing mainly on melanosomes. Despite many similarities, LROs in platelet and endothelial cells clearly possess distinct properties that allow them to provide a highly coordinated and synergistic contribution to primary hemostasis by sequentially releasing hemostatic cargo. In this brief review, we discuss in depth the known regulators of α- and δ-granules in megakaryocytes/platelets and Weibel-Palade bodies in endothelial cells, starting from transcription factors that have been associated with granule formation to protein complexes that promote granule maturation. In addition, we provide a detailed view on the interplay between platelet and endothelial LROs in controlling hemostasis as well as their dysfunction in LRO related bleeding disorders.

Melanin and other pigments are now well known to be important in exceptional preservation of soft tissues in vertebrates and other animals. Because pigments confer coloration and even structural colors, they have opened a new field of paleocolor reconstruction. Since its inception about a decade ago, reconstruction of color patterns has been performed on several vertebrates, including feathered and scale-clad dinosaurs. Iridescence and other types of structural color can also be identified through melanosome shape and arrangement. How pigments and melanosomes fossilize and are altered has become an important research subject. Ancient color patterns that may range from crypsis to brilliant displays have revealed insights into the evolution and escalation of visual systems, the nature of ancient animal interactions, and how several unique characteristics of birds already arose among dinosaurs. ▪ Melanin and other pigments preserve in exceptional fossils; this opens paths for reconstructing coloration of extinct organisms, such as dinosaurs. ▪ The most abundant pigment is melanin, which can be identified chemically and through preserved melanosome microbodies. ▪ Melanosome shape reveals clues to original hue ranging from reddish brown and black to gray and structural coloration. ▪ Other pigments may preserve, such as porphyrin pigments in theropod dinosaur eggshells. ▪ Fossil color patterns contribute new insights into the evolution of visual systems, predator-prey interactions, and key innovations.

Melanogenesis is the synthesis of the skin pigment melanin, which serves a critical role in the study of pigmentary skin diseases. Syntenin has been identified as a melanosome protein, but its role in melanogenesis is not completely understood. The present study aimed to investigate the effects and mechanisms underlying syntenin on melanogenesis in immortalized human melanocytes. Depletion of syntenin expression increased both tyrosinase (Tyr) activity and melanin content. Syntenin silencing also increased the protein expression levels of Tyr, pre-melanosomal protein and microphthalmia-associated transcription factor. In addition, the results indicated that syntenin regulated melanogenesis by upregulating the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). Taken together, these findings suggested that the regulation of melanogenesis by syntenin may be mediated by the activation of p38 MAPK and that syntenin might provide new insights into the pathogenesis of pigmented diseases.

The skin color is imparted by the pigment melanin produced in the melanosomes of melanocytes, through the catalytic action of melanogenesis enzymes tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase. Disruptions in the melanogenesis process may result to hypopigmentation, as observed in cutaneous postinflammatory conditions. Here, the bioactivity of tara tannin, specifically on melanogenesis, was evaluated in vitro using human epidermal melanocytes (HEM) and B16F10 murine melanoma cells in order to determine the possibility that it may be used as a treatment against hypopigmentation. The melanin content of tara tannin-treated B16F10 cells and the expression level of melanogenesis enzymes and melanosome transport proteins were determined. To elucidate the underlying mechanism of tara tannin’s effect on melanogenesis, DNA microarray analysis was performed. Tara tannin significantly increased melanogenesis in both murine and human pigment cell models by upregulating melanogenesis-associated enzymes’ (tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase) protein and mRNA expression levels, as well as the melanosome transport proteins (myosin Va and RAB27A) expression, both attributed to increased microphthalmia-associated transcription factor (MITF) expression. Global gene expression analysis results revealed the modulation of genes (p≤0.05; fold-change ≥2.0 and ≤−2.0) that are under the transcriptional regulation of MITF and genes relevant for MAPK signaling, metabolic pathways, and cell cycle. Tara tannin has a significant effective melanogenesis-promoting effect, making it a potential therapeutic agent against hypopigmentation disorders. This is the first report on the melanogenesis regulatory effect of tara tannin in vitro.

Tyrosinase-related protein 1 (TYRP1) is a member of tyrosinase enzyme family which is metal-containing glycoproteins localized in melanosomes where the melanin biosynthesis occurs. The downregulation of tyrosinase is the most protruding approach for the development of skin whitening and depigmentation agents, however, TYRP1 downregulation is considered a novel target for melanogenesis inhibition and is still under clinical investigations for treatment of malignant melanoma. In the present study, virtual screening using glide extra-precision docking of an in-house generated data base of 400 natural compounds belonging to family Apiaceae for TYRP1 inhibitors was performed for the first time. luteolin-7-apiosylglucoside from celery followed by 4-(beta-D-glucopyranosyloxy)benzoic acid then caffeoylquininc acid from anise showed highly potent melanogenesis inhibition even more than the synthetic standard inhibitors such as azelaic and kojic acids. These natural inhibitors exhibited good predicted skin permeability after processing with Qikprop module. Although they had slow predicted transdermal penetration rates, but these rates can be enhanced through optimizing their topical pharmaceutical formulations.

Hyperpigmentation is a skin disorder that occurs widely in the human population in which darkened patches or spots appear on the skin. Cosmetic products containing natural skin lightening agents are regarded as a safe alternative and effective approach to this hyperpigmentation problem. The review paper aims to discuss on some potential plant species with skin lightening properties by focusing on the presence of responsible bioactive compounds and their specific mechanisms of action in minimizing melanin production in hyperpigmentation problem. The finding shows that different bioactive compounds have various modes of action on melanin biosynthesis pathway including tyrosinase inhibition, melanosomes transfer inhibition, anti-oxidant and anti-inflammatory activities. Some plants with skin lightening activity contain several bioactive compounds which contribute to an enhancement of skin lightening effects and inhibition of hyperpigmentation. In conclusion, the screening of potential skin lightening ingredients from natural sources is very crucial process before formulation development for an effective skin lightening product.

Background Rab27a, Mlph, and MyoVa form a tripartite complex and relate to melanosome distribution. Melanophilin (Mlph) acts as a linker protein between Rab27a and MyoVa. The biological activity and function of 16-kauren on the expression of Mlph has not yet been studied. Objective We examined the effect of 16-kauren on melanosome transport and skin pigmentation. Methods Murine Melan-a melanocytes and SP-1 keratinocytes were used for in vitro analysis. Western blot analysis, quantitative real-time polymerase chain reaction, luciferase assay and immunohistochemical staining in 3D pigmented human skin model were performed. Results We found that 16-kauren inhibits melanosome transport in Melan-a melanocytes without affecting melanin synthesis. Treatment with 16-kauren reduced melanophilin (Mlph), a key protein in melanosome transport, in Melan-a melanocytes, at both the protein and mRNA levels while it did not affect the expression of Rab27a and MyoVa, the other two key proteins for melanosome transport. Notably, the expression of melanogenic proteins, including tyrosinase, trp1, trp2, and MITF, was not affected by 16-kauren. However, 16-kauren attenuated melanosome distribution in co-culture of Melan-a melanocytes and SP-1 keratinocytes as well as in Melan-a monolayer culture. In further confirmation of the depigmenting effects of 16-kauren on Melanoderm™, a 3D pigmented human skin model, treatment with 16-kauren for 12 days increased the brightness of the tissue as determined by lightness value and reduced the distribution of melanosomes as shown in histological examination. Conclusion These results demonstrated that 16-kauren is a selective modulator of a melangenic target, Mlph expression, and can be employed as a new depigmenting strategy.

Understanding how animal signals are produced is critical for understanding their evolution because complexity and modularity in the underlying morphology can affect evolutionary patterns. Hummingbird feathers show some of the brightest and most iridescent colors in nature. These are produced by optically complex stacks of hollow, platelet-shaped organelles called melanosomes. Neither how these morphologies produce colors nor their evolution has been systematically studied. We first used nanoscale morphological measurements and optical modeling to identify the physical basis of color production in 34 hummingbird species. We found that, in general, the melanosome stacks function as multilayer reflectors, with platelet thickness and air space size explaining variation in hue (color) and saturation (color purity). Additionally, light rays reflected from the outer keratin surface interact with those reflected by small, superficial melanosomes to cause secondary reflectance peaks, primarily in short (blue) wavelengths. We then compared variation of both the morphological components and the colors they produce. The outer keratin cortex evolves independently and is more variable than other morphological traits, possibly due to functional constraints on melanosome packing. Intriguingly, shorter wavelength colors evolve faster than longer wavelength colors, perhaps due to developmental processes that enables greater lability of the shapes of small melanosomes. Together, these data indicate that increased structural complexity of feather tissues is associated with greater variation in morphology and iridescent coloration.

Peacock feathers feature a rich gamut of colours, created by a most sophisticated structural colouration mechanism. The feather barbules contain biophotonic structures consisting of two-dimensionally-ordered lattices of cylindrical melanosomes and air channels embedded in keratin. Here, we study the reflectance characteristics of the various peacock tail feather colours by applying bifurcated-probe- and micro-spectrophotometry and imaging scatterometry. We compare the experimental results with published anatomical SEM and TEM data, using a transfer-matrix based effective-medium multilayer model that includes the number and diameter of the melanosome rodlets and air channels, the lattice spacing and the keratin cortex thickness, together with the recently determined wavelength-dependence of the refractive indices of keratin and melanin. Slight variations in the parameter values cause substantial changes in the spectral position and shape of the reflectance bands. We find that the number of layers crucially determines the number of peaks in the reflectance spectra. For a small number of melanosome layers, the reflectance band shape is particularly sensitive to the properties of the uppermost layer, which provides a simple mechanism for tuning the feather colours. This journal is

Keratinocytes uptake melanosomes from melanocytes and retain them in the perinuclear region, where they form melanin caps. Although these processes are crucial to protecting nuclear DNA against ultraviolet injury, the molecular basis of melanosome uptake and decomposition in keratinocytes is poorly understood. One of the major reasons for its being poorly understood is the lack of a specific marker protein that can be used to visualize or monitor melanosomes (or melanosome-containing compartments) that have been incorporated into keratinocytes. In this study, we performed a comprehensive localization screening for mammalian Rab family small GTPases (Rab1-45) and succeeded in identifying 11 Rabs that were enriched around melanosomes that had been incorporated into keratinocytes. We also established a new assay by using a recently developed melanosome probe (called M-INK) as a means of quantitatively assessing the degradation of proteins on incorporated melanosomes in control and each of a series of Rab-knockdown keratinocytes. The results showed that knockdown or CRISPR/Cas9-mediated knockout of Rab7B (also identified as Rab42) in keratinocytes caused strong inhibition of protein degradation on melanosomes. Our findings indicated that Rab7B/42 is recruited to melanosome-containing compartments and that it promotes protein degradation on melanosomes in keratinocytes.Key words: degradation, keratinocytes, melanocytes, melanosome, Rab small GTPase.

Prohibitin (PHB, also known as PHB1 or BAP32), is a highly conserved 31kDa protein that expressed in many cellular compartments, such as mitochondria, nucleus, cytosol, and plasma membrane, and plays roles in regulating the transcription of genes, apoptosis, and mitochondrial biogenesis. There is a report that Prohibitin expression is required for the stimulation of pigmentation by melanogenin. However, no studies have been published on the function of PHB in melanocytes, especially in melanosome transport. Methods: Immunofluorescence was performed to confirm the localization of PHB. siRNA transfections, Co-immunoprecipitation, western blotting and proximity ligation assay were performed to find binding state between proteins and demonstrate functions of PHB on melanosome transport. Results: PHB is located in the melanosome and perinuclear aggregation of melanosome is induced when expression of PHB is reduced with no influence on melanin contents. PHB binds directly to Rab27a and Mlph but not Myosin-Va. Rab27a and Mlph bind to specific domains of PHB. Reduced expression of PHB led to the impaired binding affinity between Rab27a and Mlph. Conclusion: PHB regulates melanosome transport by linking to Rab27a and Mlph in melanocytes. Targeting and regulating PHB not only manages pigmentation in melanocytes, but also controls hyperpigmentation in melanoma.

Melanin binding of drugs is known to increase drug concentrations and retention in pigmented eye tissues. Even though the correlation between melanin binding in vitro and exposure to pigmented eye in vivo has been shown, there is a discrepancy between rapid drug release from melanin particles in vitro and the long in vivo retention in the pigmented tissues. We investigated mechanisms and kinetics of pigment-related drug retention experimentally using isolated melanin particles from porcine retinal pigment epithelium and choroid, isolated porcine eye melanosomes, and re-pigmented ARPE-19 cells in a dynamic flow system. The experimental studies were supplemented with kinetic simulations. Affinity and capacity of levofloxacin, terazosin, papaverine, and timolol binding to melanin revealed Kd values of asymptotic to 50-150 mu M and B-max asymptotic to 40-112 nmol.mg(-1). The drugs were released from melanin in < 1 h (timolol) or in 6-12 h (other drugs). The drugs were released slower from the melanosomes than from melanin; the experimental differences ranged from 1.2-fold (papaverine) to 7.4-fold (timolol). Kinetic simulations supported the role of the melanosomal membrane in slowing down the release of melanin binders. In release studies from the pigmented ARPE-19 cells, drugs were released from the cellular melanin to the extra -cellular space in asymptotic to 1 day (timolol) and asymptotic to 11 days (levofloxacin), i.e., much slower than the release from melanin or melanosomes. Simulations of drug release from pigmented cells in the flow system matched the experimental data and enabled further sensitivity analyses. The simulations demonstrated a significant prolongation of drug retention in the cells as a function of decreasing drug permeability in the melanosomal membranes and increasing melanin content in the cells. Overall, we report the impact of cellular factors in prolonging drug retention and release from melanin-containing cells. These data and simulations will facilitate the design of melanin binding drugs with prolonged ocular actions.

Melanophores are pigmented cells that change the distribution of pigmented melanosomes, enabling animals to appear lighter or darker for camouflage, thermoregulation, and UV-protection. A complex series of hormonal and neural mechanisms regulates melanophore pigment distribution, making these cells a valuable tool to screen toxicants as a dynamic cell type that responds rapidly to the environment. We found that maltol, a naturally occurring flavor enhancer and fragrance agent, induces melanophore pigment aggregation in a dose-dependent manner in Xenopus laevis tadpoles. To determine if maltol affects camouflage adaptation, we placed tadpoles into maltol baths situated over either white or black background. Maltol induced pigment aggregation in a similar dose-dependent pattern regardless of background color. We also tested how maltol treatment compares to melatonin treatment and found that the degree of pigment aggregation induced by maltol is similar to treatment with melatonin, but the time course differs significantly. Last, maltol had no effect on mRNA expression of pro-opiomelanocortin or melanin concentrating hormone receptor in the brain, both of which regulate camouflage-related pigment aggregation. Our results suggest that maltol does not exert its effects via the camouflage adaptation mechanism nor via melatonin-based mechanisms. These results are the first to identify a specific toxicological effect of maltol exposure and rules out several mechanisms by which maltol may exert its effects on pigment aggregation.

Deleterious mutations within the SLC45A2 gene, encoding membrane-associated transporter protein (MATP), are responsible for type 4 oculocutaneous albinism. The cytogenetic location of SLC45A2 is 5p13.2 and it comprises seven exons located over around 40 kb. Its encoded protein, MATP, is 530 amino acids long and has 12 putative transmembrane domains. MATP is synthesized within melanocytes. It is in these cells that melanogenesis takes place and the melanin is contained within specialized organelles called melanosomes. Previous studies have shown that when MATP expression was reduced using small interfering RNA in MNT-1 melanoma cells, pH was lowered within melanosomes, they became poorly melanized and tyrosinase activity within melanocytes was also reduced. This type of albinism produces a broad spectrum of phenotypes, ranging from complete absence of melanin to brown hair and brown irides. In the current study, blood was collected from a family in which four members had oculocutaneous albinism, showing a complete absence of melanin in skin, hair and eyes. Screening of the TYR gene using the extracted DNA showed no mutation and therefore whole exome sequencing analysis was performed. A novel deletion mutation c.579delG [p.(Gly194Valfs*7)] in the SLC45A2 gene, predicted to be pathogenic and to result in both frameshift and premature termination of the MATP chain, was identified. These data add to the information pertaining to the mutation spectrum of OCA4.