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MFSD12 protein has recently risen as a key factor in malignancy and plays a potential role in a variety of complex oncogenic signaling cascades. Current studies suggest that MFSD12 has a positive complex role in the growth and progression of tumors such as melanoma, breast cancer, and lung cancer. At the same time, as a transporter of cysteine, MFSD12 is also involved in the development of lysosomal storage diseases. Therefore, MFSD12 may be an effective target to inhibit tumor development, block metastasis, and expand the therapeutic effect. This article reviews the molecular mechanisms of MFSD12 in a variety of cancers and lysosomal storage diseases. [ABSTRACT FROM AUTHOR]

Human pigmentary disorders encompass a broad spectrum of phenotypic changes arising from disruptions in various stages of melanocyte formation, the melanogenesis process, or the transfer of pigment from melanocytes to keratinocytes. A large number of pigmentation genes associated with pigmentary disorders have been identified, many of them awaiting in vivo confirmation. A more comprehensive understanding of the molecular basis of pigmentary disorders requires a vertebrate animal model where changes in pigmentation are easily observable in vivo and can be combined to genomic modifications and gain/loss‐of‐function tools. Here we present the amphibian Xenopus with its unique features that fulfill these requirements. Changes in pigmentation are particularly easy to score in Xenopus embryos, allowing whole‐organism based phenotypic screening. The development and behavior of Xenopus melanocytes closely mimic those observed in mammals. Interestingly, both Xenopus and mammalian skins exhibit comparable reactions to ultraviolet radiation. This review highlights how Xenopus constitutes an alternative and complementary model to the more commonly used mouse and zebrafish, contributing to the advancement of knowledge in melanocyte cell biology and related diseases. [ABSTRACT FROM AUTHOR]

Melasma is a common condition of hyperpigmented facial skin. Picosecond lasers are reported to be effective for the treatment of melasma. We aimed to identify the most effective therapeutic mode and elucidate the potential molecular mechanisms of picosecond lasers for the treatment of melasma. Female Kunming mice with melasma‐like conditions were treated using four different picosecond laser modes. Concurrently, in vitro experiments were conducted to assess changes in melanin and autophagy in mouse melanoma B16‐F10 cells treated with these laser modes. Changes in melanin in mouse skin were detected via Fontana–Masson staining, and melanin particles were evaluated in B16‐F10 cells. Real‐time polymerase chain reaction and western blotting were used to analyse the expression levels of melanosome and autophagy‐related messenger ribonucleic acid (mRNA) and proteins. A combination of large‐spot low‐fluence 1064‐nm and fractional 1064‐nm picosecond lasers resulted insignificant decreases in melanin as well as in mRNA and protein expression of melanin‐synthesizing enzymes (TYR, TRP‐1 and MITF). This combination also led to increased expression of the autophagy‐related proteins, Beclin1 and ATG5, with a marked decrease in p62 expression. Intervention with the PI3K activator, 740 Y‐P, increased TYR, TRP‐1, MITF, p‐PI3K, p‐AKT, p‐mTOR and p62 expression but decreased the expression of LC3, ATG5 and Beclin1. A combination of large‐spot low‐fluence 1064‐nm and fractional 1064‐nm picosecond lasers proved more effective and safer. It inhibits melanin production, downregulates the PI3K/AKT/mTOR pathway, enhances melanocyte autophagy and accelerates melanin metabolism, thereby reducing melanin content. [ABSTRACT FROM AUTHOR]

Melanosomes are specific organelles dedicated to melanin synthesis and accumulation in melanocytes. Autophagy is suggestively involved in melanosome degradation, although the potential underlying molecular mechanisms remain elusive. In selective autophagy, autophagy receptors and E3-ligases are the key factors conferring cargo selectivity. In B16F10 cells, β-mangostin efficiently induced melanosome degradation without affecting other organelles such as mitochondria, peroxisomes, and the endoplasmic reticulum. Among various autophagy receptors, optineurin (OPTN) contributes TANK-binding kinase 1 (TBK1)-dependently to melanosome degradation and its knockdown inhibited β-mangostin-mediated melanosome degradation. OPTN translocation to melanosomes was dependent on its ubiquitin-binding domain. Moreover, OPTN-mediated TBK1 activation and subsequent TBK1-mediated S187 OPTN phosphorylation were essential for melanosome degradation. β-mangostin increased K63-linked melanosome ubiquitination. Finally, the E3-ligase RCHY1 knockdown inhibited the melanosome ubiquitination required for OPTN- and TBK1-phosphorylation as well as melanosome degradation. This study suggests that melanophagy, melanosome-selective autophagy, contributes to melanosome degradation, and OPTN and RCHY1 are an essential autophagy receptor and a E3-ligase, respectively, conferring cargo selectivity in melanophagy. [ABSTRACT FROM AUTHOR]

A specialized membrane-bound organelle, named the melanosome, is central to the storage and transport of melanin as well as melanin synthesis in melanocytes. Although previous studies have linked melanosomal degradation to autophagy, the precise mechanisms remain elusive. Autophagy, a complex catabolic process involving autophagosomes and lysosomes, plays a vital role in cellular constituent degradation. In this study, the role of autophagy in melanosomal degradation was explored, employing a cell-based screening system designed to unveil key pathway regulators. We identified specific dipeptidyl peptidase-4 inhibitors, such as teneligliptin hydrobromide and retagliptin phosphate, as novel agents inducing melanophagy through a comprehensive screening of a ubiquitination-related chemical library. We found that treatment with teneligliptin hydrobromide or retagliptin phosphate not only diminishes melanin content elevated by alpha-melanocyte-stimulating hormone (α-MSH) but also triggers autophagy activation within B16F1 cells. In addition, the targeted inhibition of unc-51-like kinase (ULK1) significantly attenuated both the anti-pigmentation effects and autophagy induced by teneligliptin hydrobromide and retagliptin phosphate in α-MSH-treated cells. Collectively, our data demonstrate a new frontier in understanding melanosomal degradation, identifying teneligliptin hydrobromide and retagliptin phosphate as promising inducers of melanophagy via autophagy activation. This study contributes essential insights into cellular degradation mechanisms and offers potential therapeutic avenues in the regulation of pigmentation. [ABSTRACT FROM AUTHOR]

Epimedin B (EB) is one of the main flavonoid ingredients present in Epimedium brevicornum Maxim., a traditional herb widely used in China. Our previous study showed that EB was a stronger inducer of melanogenesis and an activator of tyrosinase (TYR). However, the role of EB in melanogenesis and the mechanism underlying the regulation remain unclear. Herein, as an extension to our previous investigation, we provide comprehensive evidence of EB-induced pigmentation in vivo and in vitro and elucidate the melanogenesis mechanism by assessing its effects on the TYR family of proteins (TYRs) in terms of expression, activity, and stability. The results showed that EB increased TYRs expression through microphthalmia-associated transcription factor-mediated p-Akt (referred to as protein kinase B (PKB))/glycogen synthase kinase 3β (GSK3β)/β-catenin, p-p70 S6 kinase cascades, and protein 38 (p38)/mitogen-activated protein (MAP) kinase (MAPK) and extracellular regulated protein kinases (ERK)/MAPK pathways, after which EB increased the number of melanosomes and promoted their maturation for melanogenesis in melanoma cells and human primary melanocytes/skin tissues. Furthermore, EB exerted repigmentation by stimulating TYR activity in hydroquinone- and N-phenylthiourea-induced TYR inhibitive models, including melanoma cells, zebrafish, and mice. Finally, EB ameliorated monobenzone-induced depigmentation in vitro and in vivo through the enhancement of TYRs stability by inhibiting TYR misfolding, TYR-related protein 1 formation, and retention in the endoplasmic reticulum and then by downregulating the ubiquitination and proteolysis processes. These data conclude that EB can target TYRs and alter their expression, activity, and stability, thus stimulating their pigmentation function, which might provide a novel rational strategy for hypopigmentation treatment in the pharmaceutical and cosmetic industries.

Brown-and-white giant pandas (hereafter brown pandas) are distinct coat color mutants found exclusively in the Qinling Mountains, Shaanxi, China. However, its genetic mechanism has remained unclear since their discovery in 1985. Here, we identified the genetic basis for this coat color variation using a combination of field ecological data, population genomic data, and a CRISPR--Cas9 knockout mouse model. We de novo assembled a long-read-based giant panda genome and resequenced the genomes of 35 giant pandas, including two brown pandas and two family trios associated with a brown panda. We identified a homozygous 25-bp deletion in the first exon of Bace2, a gene encoding amyloid precursor protein cleaving enzyme, as the most likely genetic basis for brown-and-white coat color. This deletion was further validated using PCR and Sanger sequencing of another 192 black giant pandas and CRISPR-Cas9 edited knockout mice. Our investigation revealed that this mutation reduced the number and size of melanosomes of the hairs in knockout mice and possibly in the brown panda, further leading to the hypopigmentation. These findings provide unique insights into the genetic basis of coat color variation in wild animals. [ABSTRACT FROM AUTHOR]

Objective Methods Results Conclusion Tyrosinase inhibitors suppress melanogenesis in melanocytes. During a screening for tyrosinase inhibitors, however, we noticed some discrepancies in inhibitory efficacies between melanocytes and in vitro assays. The compound (S)‐N‐{3‐[4‐(dimethylamino)phenyl]propyl}‐N‐methyl‐indan‐1‐amine (GIF‐2115) exerts antioxidative stress activity upon accumulation in late endosomes and lysosomes. GIF‐2115 was also identified as a potent antimelanogenic reagent in B16F10 mouse melanoma cells. GIF‐2115 inhibited the activity of mushroom tyrosinase and the lysates of B16F10 cells. However, structure–activity relationship studies indicated that GIF‐2238, which lacks the benzene ring in the aminoindan structure of GIF‐2115, inhibited tyrosinase activity in vitro but did not inhibit melanogenesis in B16F10 cells. The aim of the present study is to show the importance of the intracellular distribution of tyrosinase inhibitors in exerting their antimelanogenic activity in melanocytes.The intracellular distribution of compounds was monitored by linking with the fluorescent group of 7‐nitro‐2,1,3‐benzoxadiazole (NBD). To mislocalize GIF‐2115 to mitochondria, the mitochondria‐preferring fluoroprobe ATTO565 was used.We reconfirmed the localization of GIF‐2250 (GIF‐2115‐NBD) not only to matured but also to early‐stage melanosomes. Although GIF‐2286 (GIF‐2238‐NBD) maintained tyrosinase inhibitory activity, it did not show specific intracellular localization. Moreover, when GIF‐2115 was linked with ATTO565, the resultant compound GIF‐2265 did not inhibit melanogenesis in B16F10 cells, despite its strong tyrosinase inhibitory activity.These results suggest that melanosomal localization is essential for the antimelanogenic activity of GIF‐2115, and GIF‐2115 derivatives may be new guides for drugs to endosomes and lysosomes as well as melanosomes. [ABSTRACT FROM AUTHOR]

Epimedin B (EB) is one of the main flavonoid ingredients present in Epimedium brevicornum Maxim., a traditional herb widely used in China. Our previous study showed that EB was a stronger inducer of melanogenesis and an activator of tyrosinase (TYR). However, the role of EB in melanogenesis and the mechanism underlying the regulation remain unclear. Herein, as an extension to our previous investigation, we provide comprehensive evidence of EB-induced pigmentation in vivo and in vitro and elucidate the melanogenesis mechanism by assessing its effects on the TYR family of proteins (TYRs) in terms of expression, activity, and stability. The results showed that EB increased TYRs expression through microphthalmia-associated transcription factor-mediated p-Akt (referred to as protein kinase B (PKB))/glycogen synthase kinase 3β (GSK3β)/β-catenin, p-p70 S6 kinase cascades, and protein 38 (p38)/mitogen-activated protein (MAP) kinase (MAPK) and extracellular regulated protein kinases (ERK)/MAPK pathways, after which EB increased the number of melanosomes and promoted their maturation for melanogenesis in melanoma cells and human primary melanocytes/skin tissues. Furthermore, EB exerted repigmentation by stimulating TYR activity in hydroquinone- and N -phenylthiourea-induced TYR inhibitive models, including melanoma cells, zebrafish, and mice. Finally, EB ameliorated monobenzone-induced depigmentation in vitro and in vivo through the enhancement of TYRs stability by inhibiting TYR misfolding, TYR-related protein 1 formation, and retention in the endoplasmic reticulum and then by downregulating the ubiquitination and proteolysis processes. These data conclude that EB can target TYRs and alter their expression, activity, and stability, thus stimulating their pigmentation function, which might provide a novel rational strategy for hypopigmentation treatment in the pharmaceutical and cosmetic industries. [Display omitted] • It is first time to investigate and determine the pharmacological activity of Epimedin B in pigment synthesis. • Multiple depigmented models including in vivo and in vitro are utilized to evaluate effect of Epimedin B in pigment synthesis. • Epimedin B can target TYRs in terms of three aspects (expression, activity, and stability) to exert pigmentation function. [ABSTRACT FROM AUTHOR]

Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, we identify a third interaction between Myo5a and Mlph, that is, the interaction between the exon-G-encoded region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a transportation of melanosomes.

Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein's fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called "amyloidosis". Preclinical animal models have failed to model this PMEL "dysamyloidosis" pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL's repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein's repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL's repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease. [ABSTRACT FROM AUTHOR]

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Colour reconstructions have provided new insights into the lives of dinosaurs and other extinct animals, by predicting colouration patterns from fossilised pigment-bearing organelles called melanosomes. Although these methods have become increasingly popular, only a small number of dinosaurs have been studied using these techniques, which require exceptional preservation of fossil feathers, leaving open key questions such as whether dinosaurs changed their plumage patterns during ontogeny. Here we reconstruct the feather colouration of an approximately one-year-old individual of the Early Cretaceous dromaeosaurid theropod Wulong bohaiensis, which to our knowledge is the first unequivocal juvenile paravian for which aspects of the original colour has been predicted. Using quadratic discriminant analysis (QDA) and multinomial logistic regression (MLR) on the most comprehensive available datasets, we find strong evidence for iridescent plumage of the forelimb and hindlimb remiges and grey plumage on other portions of the body. This suggests that some juvenile paravians used shiny iridescent feathers for signalling purposes, possibly even before reaching somatic or sexual maturity, and thus we can conclude that this paravian used iridescent signalling for intraspecific communication other than sexual signalling. Finally, our results show that when analysing fossil datasets that are entirely comprised of solid and cylindrical melanosomes QDA consistently outperforms MLR, providing more accurate and higher classification probability colour predictions.

The deep-sea environment is associated with a wide range of anatomically specialized morphologies allowing camouflage in this low or no light environment. Specialized ultra-black coloration has been documented in the pelican eel, Eurypharynx pelecanoides, but has not been explored in the other largely deep-sea inhabiting pelagic anguilloid eels. Histological examination of the integument revealed a layer of free melanosomes in the superficial dermis consistent with specialized ultra-black camouflage in the swallower eels Saccopharynx, the bobtail snipe eel Cyema, the sawtooth eels Serrivomer, and the snipe eels Avocettina and Nemichthys. The anatomy in these taxa is consistent with the previously described ultra-black morphology, except that Nemichthys, Avocettina, and Serrivomer have both large amounts of free melanosomes and melanophores. Consideration of this morphology in the context of anguilloid eel evolution in the deep-sea environment suggests repeated independent evolution of ultra-black coloration within the anguilloids, and greater development in the taxa more specifically associated with the bathypelagic habitats and the production of bioluminescence. [ABSTRACT FROM AUTHOR]

Skin pigmentation ensures efficient photoprotection and relies on the pigment melanin, which is produced by epidermal melanocytes and transferred to surrounding keratinocytes. While the molecular mechanisms of melanin synthesis and transport in melanocytes are now well characterized, much less is known about melanin transfer and processing within keratinocytes. Over the past few decades, distinct models have been proposed to explain how melanin transfer occurs at the cellular and molecular levels. However, this remains a debated topic, as up to four different models have been proposed, with evidence presented supporting each. Here, we review the current knowledge on the regulation of melanin exocytosis, internalization, processing, and polarization. Regarding the different transfer models, we discuss how these might co-exist to regulate skin pigmentation under different conditions, i.e., constitutive and facultative skin pigmentation or physiological and pathological conditions. Moreover, we discuss recent evidence that sheds light on the regulation of melanin exocytosis by melanocytes and internalization by keratinocytes, as well as how melanin is stored within these cells in a compartment that we propose be named the melanokerasome. Finally, we review the state of the art on the molecular mechanisms that lead to melanokerasome positioning above the nuclei of keratinocytes, forming supranuclear caps that shield the nuclear DNA from UV radiation. Thus, we provide a comprehensive overview of the current knowledge on the molecular mechanisms regulating skin pigmentation, from melanin exocytosis by melanocytes and internalization by keratinocytes to processing and polarization within keratinocytes. A better knowledge of these molecular mechanisms will clarify long-lasting questions in the field that are crucial for the understanding of skin pigmentation and can shed light on fundamental aspects of organelle biology. Ultimately, this knowledge can lead to novel therapeutic strategies to treat hypo- or hyper-pigmentation disorders, which have a high socio-economic burden on patients and healthcare systems worldwide, as well as cosmetic applications. [ABSTRACT FROM AUTHOR]

Background: Melanosomes are lysosome-related organelles that contain melanogenic factors and synthesize melanin as they mature. FYVE finger-containing phosphoinositide kinase (PIKfyve) regulates late endosome and lysosome morphology, vesicle trafficking, and autophagy. In melanocytes, PIKfyve inhibition has been reported to induce hypopigmentation due to impairments in the metabolism of early-stage melanosomes. Methods and results: Here, we report a new type of melanosome metabolism: post-PIKfyve inhibition, which was found during the characterization of the endosome/lysosome fluoroprobe GIF-2250. In B16F10 mouse melanoma cells, GIF-2250 highlighted vesicles positive for lysosomal-associated membrane protein 1 (lysosome marker) and other endosome/lysosome markers (CD63 and Rab7/9). When cells were continuously treated with PIKfyve inhibitors, intracellular vacuoles formed, while GIF-2250 fluorescence signals diminished and were diffusely distributed in the vacuoles. After removal of the PIKfyve inhibitors, the GIF-2250 signal intensity was restored, and some GIF-2250-positive vesicles wrapped the melanosomes, which spun at high speed. In addition, intermittent PIKfyve inhibition caused melanin diffusion in the vacuoles and possible leakage into the cytoplasmic compartments, and melanosome degradation was detected by a transmission electron microscope. Melanosome degradation was accompanied by decreased levels of melanin synthesis enzymes and increased levels of the autophagosome maker LC3BII, which is also associated with early melanosomes. However, the protein levels of p62, which is degraded during autophagy, were increased, suggesting an impairment in autophagy flux during intermittent PIKfyve inhibition. Moreover, the autophagy inhibitor 3-methyladenine does not affect these protein levels, suggesting that the melanosome degradation by the intermittent inhibition of PIKfyve is not mediated by canonical autophagy. Conclusions: In conclusion, disturbance of PIKfyve activity induces melanosome degradation in a canonical autophagy-independent manner. [ABSTRACT FROM AUTHOR]

Melanins are the main pigments found in mammals. Their synthesis and transfer to keratinocytes have been widely investigated for many years. However, analysis has been mainly carried out using fixed rather than live cells. In this study, we have analysed the melanosomes in living mammalian cells using newly developed scanning electron-assisted dielectric microscopy (SE-ADM). The melanosomes in human melanoma MNT-1 cells were observed as clear black particles in SE-ADM. The main structure of melanosomes was toroidal while that of normal melanocytes was ellipsoidal. In tyrosinase knockout MNT-1 cells, not only the black particles in the SE-ADM images but also the Raman shift of melanin peaks completely disappeared suggesting that the black particles were really melanosomes. We developed a deep neural network (DNN) system to automatically detect melanosomes in cells and analysed their diameter and roundness. In terms of melanosome morphology, the diameter of melanosomes in melanoma cells did not change while that in normal melanocytes increased during culture. The established DNN analysis system with SE-ADM can be used for other particles, e.g. exosomes, lysosomes, and other biological particles.

A specialized membrane-bound organelle, named the melanosome, is central to the storage and transport of melanin as well as melanin synthesis in melanocytes. Although previous studies have linked melanosomal degradation to autophagy, the precise mechanisms remain elusive. Autophagy, a complex catabolic process involving autophagosomes and lysosomes, plays a vital role in cellular constituent degradation. In this study, the role of autophagy in melanosomal degradation was explored, employing a cell-based screening system designed to unveil key pathway regulators. We identified specific dipeptidyl peptidase-4 inhibitors, such as teneligliptin hydrobromide and retagliptin phosphate, as novel agents inducing melanophagy through a comprehensive screening of a ubiquitination-related chemical library. We found that treatment with teneligliptin hydrobromide or retagliptin phosphate not only diminishes melanin content elevated by alpha-melanocyte-stimulating hormone (α-MSH) but also triggers autophagy activation within B16F1 cells. In addition, the targeted inhibition of unc-51-like kinase (ULK1) significantly attenuated both the anti-pigmentation effects and autophagy induced by teneligliptin hydrobromide and retagliptin phosphate in α-MSH-treated cells. Collectively, our data demonstrate a new frontier in understanding melanosomal degradation, identifying teneligliptin hydrobromide and retagliptin phosphate as promising inducers of melanophagy via autophagy activation. This study contributes essential insights into cellular degradation mechanisms and offers potential therapeutic avenues in the regulation of pigmentation.

Lysosomal degradation of tyrosinase, a pivotal enzyme in melanin synthesis, negatively impacts melanogenesis in melanocytes. Nevertheless, the precise molecular mechanisms by which lysosomes target tyrosinase have remained elusive. Here, we identify RING (Really Interesting New Gene) finger protein 152 (RNF152) as a membrane-associated ubiquitin ligase specifically targeting tyrosinase for the first time, utilizing AlphaScreen technology. We observed that modulating RNF152 levels in B16 cells, either via overexpression or siRNA knockdown, resulted in decreased or increased levels of both tyrosinase and melanin, respectively. Notably, RNF152 and tyrosinase co-localized at the trans-Golgi network (TGN). However, upon treatment with lysosomal inhibitors, both proteins appeared in the lysosomes, indicating that tyrosinase undergoes RNF152-mediated lysosomal degradation. Through ubiquitination assays, we found the indispensable roles of both the RING and transmembrane (TM) domains of RNF152 in facilitating tyrosinase ubiquitination. In summary, our findings underscore RNF152 as a tyrosinase-specific ubiquitin ligase essential for regulating melanogenesis in melanocytes.

Objective: Various histological studies of facial pigmented spot sites such as solar lentigo have been reported, but few studies have used quantitative indices by histomorphometric analysis of the internal structure of pigmented spot sites using non‐invasive methods. In the present study, to quantitatively elucidate morphological changes in the epidermis in male, darker‐pigmented spots and female, light‐pigmented spots, indices that characterize the internal structure of the epidermis in pigmented spot sites were measured using in vivo confocal laser scanning microscopy (CLSM). Methods: The darkness of pigmented spots on the cheeks of 69 women and 43 men was analysed using image analysis software. The L* value was calculated from RGB values obtained from facial images. The internal structures of pigmented spots on the cheeks of 13 subjects were observed by CLSM. Various parameters were measured using CLSM images from the surface of the stratum corneum to the bottom of the dermal papillae, including the thickness of the epidermis, melanosome content, and shape of the dermal papillae. Results: Mean ΔL* values between pigmented spots and non‐pigmented areas of male subjects were significantly increased in the 40s and 50s compared with those of female subjects. Conspicuous pigmented spots increased in the 40s in male subjects and the 50s in female subjects. In CLSM observations, significant increases in the thickness of the epidermis and melanosome content were confirmed in pigmented spots compared with surrounding non‐pigmented areas. In particular, melanosome content in the male subject group with dark‐coloured pigmented spots increased significantly to about eight times that of non‐pigmented areas, and more than double that of the male subject group with light‐coloured pigmented spots. Conclusion: From the measurements of quantitative parameters, morphological changes in the epidermis were clearly related to the surface colour tone of pigmented spots. Darker pigmented spot sites tended to show longer rete pegs in the epidermis. Accumulation of melanosomes in epidermal basal cells could be considered to increase with the degree of elongation of rete pegs at pigmented spot sites and, thus, induce darker pigmented spots. [ABSTRACT FROM AUTHOR]

The color of Mollusca shells is one of the most important attributes to consumers. At the cellular level, black color is mainly from the melanin produced by melanocytes. The melanosome is a specialized membrane-bound organelle that is involved in melanin synthesis, storage, and transportation. How the complex pigmentation process in the Crassostrea gigas is established remains an open question. The objectives of this studies are to examine the morphological characteristics of melanosomes or melanin of mantle pigmentation in the Pacific oyster, thereby investigating its contribution to shell color. The results show that pigmented granules of the mantles vary among the three lobes, and the melanosomes at different stages are enriched in distinct cargo molecules, which indicate the remarkable difference between the marginal mantle and central mantle. Examination of mantle histology reveals that the mantle margin of the oyster is characterized by three different folds, including the outer secretory, middle sensory, and inner muscular fold. Ferrous ion chelating assays against the tyrosine hydroxylase indicate that a large amount of melanin is localized in the inner surface of the middle fold. Transmission electron microscopy analyses show that the mantle edge is composed of tall columnar and cuboidal epidermal cells and some pigmented melanocytes intersperse among these cells. The numbers of melanosomes among the three lobes are different. In the inner fold and the middle fold of the mantle, some single dispersion, or aggregation of melanosomes with different degrees of melanization are found in the outer surface. Numerous melanosomes are distributed in the epithelium of the outer fold of the mantle, and mainly are at the apical microvillar surface near the lumen. However, melanosomes are occasionally observed in the central mantle, and they are relatively less. This work provides new insights into the process of melanin deposit in the mantle and shell pigmentation in C. gigas. [ABSTRACT FROM AUTHOR]

Colour reconstructions have provided new insights into the lives of dinosaurs and other extinct animals, by predicting colouration patterns from fossilised pigment-bearing organelles called melanosomes. Although these methods have become increasingly popular, only a small number of dinosaurs have been studied using these techniques, which require exceptional preservation of fossil feathers, leaving open key questions such as whether dinosaurs changed their plumage patterns during ontogeny. Here we reconstruct the feather colouration of an approximately one-year-old individual of the Early Cretaceous dromaeosaurid theropod Wulong bohaiensis, which to our knowledge is the first unequivocal juvenile paravian for which aspects of the original colour has been predicted. Using quadratic discriminant analysis (QDA) and multinomial logistic regression (MLR) on the most comprehensive available datasets, we find strong evidence for iridescent plumage of the forelimb and hindlimb remiges and grey plumage on other portions of the body. This suggests that some juvenile paravians used shiny iridescent feathers for signalling purposes, possibly even before reaching somatic or sexual maturity, and thus we can conclude that this paravian used iridescent signalling for intraspecific communication other than sexual signalling. Finally, our results show that when analysing fossil datasets that are entirely comprised of solid and cylindrical melanosomes QDA consistently outperforms MLR, providing more accurate and higher classification probability colour predictions. [ABSTRACT FROM AUTHOR]

Serotonin (5‐HT), a neurotransmitter, is essential for normal and pathological pigmentation processing, and its receptors may be therapeutical targets. The effect and behavior of the 5‐HT7 receptor (5‐HT7R) in melanogenesis in high vertebrates remain unknown. Herein, we examine the role and molecular mechanism of 5‐HT7R in the pigmentation of human skin cells, human tissue, mice, and zebrafish models. Firstly, 5‐HT7R protein expression decreased significantly in stress‐induced depigmentation skin and vitiligo epidermis. Stressed mice received transdermal serotonin 5‐HT7R selective agonists (LP‐12, 0.01%) for 12 or 60 days. Mice might recover from persistent stress‐induced depigmentation. The downregulation of tyrosinase (Tyr), microphthalmia‐associated transcription factor (Mitf) expression, and 5‐HT7R was consistently restored in stressed skin. High‐throughput RNA sequencing showed that structural organization (dendrite growth and migration) and associated pathways were activated in the dorsal skin of LP‐12‐treated animals. 5‐HT7R selective agonist, LP‐12, had been demonstrated to enhance melanin production, dendrite growth, and chemotactic motility in B16F10 cells, normal human melanocytes (NHMCs), and zebrafish. Mechanistically, the melanogenic, dendritic, and migratory functions of 5‐HT7R were dependent on the downstream signaling of cAMP‐PKA‐ERK1/2, JNK MAPK, RhoA/Rab27a, and PI3K/AKT pathway activation. Importantly, pharmacological inhibition and genetic siRNA of 5‐HT7R by antagonist SB269970 partially/completely abolished these functional properties and the related activated pathways in both NHMCs and B16F10 cells. Consistently, htr7a/7b genetic knockdown in zebrafish could blockade melanogenic effects and abrogate 5‐HT‐induced melanin accumulation. Collectively, we have first identified that 5‐HT7R regulates melanogenesis, which may be a targeted therapy for pigmentation disorders, especially those worsened by stress. [ABSTRACT FROM AUTHOR]

Melanin is synthesized in melanocytes and is transferred into keratinocytes to block the effects of ultraviolet (UV) radiation and is important for preventing skin cancers including melanoma. However, it is known that after melanomagenesis and melanoma invasion or metastases, melanin synthesis still occurs. Since melanoma cells are no longer involved in the sun tanning process, it is unclear why melanocytes would maintain melanin synthesis after melanomagenesis has occurred. Aside from blocking UV-induced DNA mutation, melanin may provide other metabolic functions that could benefit melanoma. In addition, studies have suggested that there may be a selective advantage to melanin synthesis in melanoma; however, mechanisms regulating melanin synthesis outside the epidermis or hair follicle is unknown. We will discuss how melanosomal pH controls melanin synthesis in melanocytes and how melanosomal pH control of melanin synthesis might function in melanoma. We will also discuss potential reasons why melanin synthesis might be beneficial for melanoma cellular metabolism and provide a rationale for why melanin synthesis is not limited to benign melanocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

B16-F1 melanoma cells have often been used as a model to investigate melanogenesis, but the evidence that melanosome biogenesis and transport occur by the same mechanisms in normal melanocytes and B16-F1 cells is insufficient. In this study, we established knockout B16-F1 cells for each of several key factors in melanogenesis, i.e., tyrosinase (Tyr), Hps4, Rab27A, and Rab32·Rab38 (Rab32/38), and then compared their phenotypes with the phenotypes of corresponding mutant mouse melanocyte cell lines, i.e., melan-c, melan-le, melan-ash, and Rab32-deficient melan-cht cells, respectively. The results showed that Tyr and Rab27A are also indispensable for melanin synthesis and peripheral melanosome distribution, respectively, in B16-F1 cells, but that Hps4 or its downstream targets Rab32/38 are not essential for Tyr transport in B16-F1 cells, suggesting the existence of a Rab32/38-independent Tyr transport mechanism in B16-F1 cells. We then performed comprehensive knockdown screening of Rab small GTPases and identified Rab10 and Rab24, previously uncharacterized Rabs in melanocytes, as being involved in Tyr transport under Rab32/38-null conditions. Our findings indicate a difference between the Tyr transport mechanism in melanocytes and B16-F1 cells in terms of Rab32/38-dependency and a limitation in regard to using melanoma cells as a model for melanocytes, especially when investigating the mechanism of endosomal Tyr transport. [ABSTRACT FROM AUTHOR]

Hermansky-Pudlak syndrome (HPS) is an inherited disorder of intracellular vesicle trafficking affecting the function of lysosome-related organelles (LROs). At least 11 genes underlie the disease, encoding four protein complexes, of which biogenesis of lysosome-related organelles complex-2 (BLOC-2) is the last whose molecular action is unknown. We find that the unicellular eukaryote Dictyostelium unexpectedly contains a complete BLOC-2, comprising orthologs of the mammalian subunits HPS3, -5, and -6, and a fourth subunit, an ortholog of the Drosophila LRO-biogenesis gene, Claret. Lysosomes from Dictyostelium BLOC-2 mutants fail to mature, similar to LROs from HPS patients, but for all endolysosomes rather than a specialized subset. They also strongly resemble lysosomes from WASH mutants. Dictyostelium BLOC-2 localizes to the same compartments as WASH, and in BLOC-2 mutants, WASH is inefficiently recruited, accounting for their impaired lysosomal maturation. BLOC-2 is recruited to endolysosomes via its HPS3 subunit. Structural modeling suggests that all four subunits are proto-coatomer proteins, with important implications for BLOC-2's molecular function. The discovery of Dictyostelium BLOC-2 permits identification of orthologs throughout eukaryotes. BLOC-2 and lysosome-related organelles, therefore, pre-date the evolution of Metazoa and have broader and more conserved functions than previously thought. [Display omitted] • Dictyostelium BLOC-2 comprises orthologs of the mammalian subunits, plus Claret • BLOC-2 mutants have disrupted endolysosome maturation, leading to delayed exocytosis • BLOC-2 functions at the same compartment as WASH, but genetically upstream • BLOC-2 subunit genes are found in diverse eukaryotes but may have been widely lost Thomason et al. discover that biogenesis of lysosome-related organelles complex-2 (BLOC-2) is an ancient proto-coatomer complex widely conserved throughout eukaryotes. In the unicellular Dictyostelium , it functions in a central endolysosomal vesicle maturation pathway, perhaps reflective of its role in the last eukaryotic common ancestor. [ABSTRACT FROM AUTHOR]

Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein’s fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called “amyloidosis”. Preclinical animal models have failed to model this PMEL “dysamyloidosis” pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL’s repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein’s repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL’s repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease.

Skin pigmentation ensures efficient photoprotection and relies on the pigment melanin, which is produced by epidermal melanocytes and transferred to surrounding keratinocytes. While the molecular mechanisms of melanin synthesis and transport in melanocytes are now well characterized, much less is known about melanin transfer and processing within keratinocytes. Over the past few decades, distinct models have been proposed to explain how melanin transfer occurs at the cellular and molecular levels. However, this remains a debated topic, as up to four different models have been proposed, with evidence presented supporting each. Here, we review the current knowledge on the regulation of melanin exocytosis, internalization, processing, and polarization. Regarding the different transfer models, we discuss how these might co-exist to regulate skin pigmentation under different conditions, i.e., constitutive and facultative skin pigmentation or physiological and pathological conditions. Moreover, we discuss recent evidence that sheds light on the regulation of melanin exocytosis by melanocytes and internalization by keratinocytes, as well as how melanin is stored within these cells in a compartment that we propose be named the melanokerasome. Finally, we review the state of the art on the molecular mechanisms that lead to melanokerasome positioning above the nuclei of keratinocytes, forming supranuclear caps that shield the nuclear DNA from UV radiation. Thus, we provide a comprehensive overview of the current knowledge on the molecular mechanisms regulating skin pigmentation, from melanin exocytosis by melanocytes and internalization by keratinocytes to processing and polarization within keratinocytes. A better knowledge of these molecular mechanisms will clarify long-lasting questions in the field that are crucial for the understanding of skin pigmentation and can shed light on fundamental aspects of organelle biology. Ultimately, this knowledge can lead to novel therapeutic strategies to treat hypo- or hyper-pigmentation disorders, which have a high socio-economic burden on patients and healthcare systems worldwide, as well as cosmetic applications.

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.

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 K d values of ≈ 50–150 μM and B max ≈ 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 extracellular space in ≈ 1 day (timolol) and ≈ 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Simple Summary: Melanoma is produced by the malignant transformation of the pigmented cells in the skin. It is the deadliest form of skin cancer and a global medical burden. Recent research suggests that extracellular vesicles improve diagnosis and treatment of melanoma. Extracellular vesicles are virus-like vehicles that are released into the blood and other body fluids by most cell types, including cancer cells. They sequester molecular substances from the cytoplasm and transport them as messengers to target cells. Thanks to these properties, extracellular vesicles provide a molecular fingerprint of the cell of origin and can serve as biomarkers for cancer diagnosis or prognosis. In addition, molecular signals exchanged through extracellular vesicles between cancer cells and the tumor environment can reveal signaling pathways that are important for cancer progression. In this review we give a general overview of extracellular vesicles and focus on their impact on melanoma progression and potential use as biomarkers for monitoring and treating melanoma. Cutaneous melanoma arises from a malignant transformation of the melanocytes in the skin. It is the deadliest form of skin cancer owing to its potential to metastasize. While recent advances in immuno-oncology have been successful in melanoma treatment, not all the patients respond to the treatment equally, thus individual pre-screening and personalized combination therapies are essential to stratify and monitor patients. Extracellular vesicles (EVs) have emerged as promising biomarker candidates to tackle these challenges. EVs are ~50–1000-nm-sized, lipid bilayer-enclosed spheres, which are secreted by almost all cell types, including cancer cells. Their cargo, such as nucleic acids, proteins, lipids, amino acids, and metabolites, can be transferred to target cells. Thanks to these properties, EVs can both provide a multiplexed molecular fingerprint of the cell of origin and thus serve as potential biomarkers, or reveal pathways important for cancer progression that can be targeted pharmaceutically. In this review we give a general overview of EVs and focus on their impact on melanoma progression. In particular, we shed light on the role of EVs in shaping the tumor–stroma interactions that facilitate metastasis and summarize the latest findings on molecular profiling of EV-derived miRNAs and proteins that can serve as potential biomarkers for melanoma progression. [ABSTRACT FROM AUTHOR]

Keratinocytes are recipients of melanosomes. Although the chemical basis of melanogenesis is well documented, the molecular mechanism of melanosome transfer must be elucidated. TRPA1 is a member of the transient receptor potential A subfamily. Previous studies have shown that inhibition of TRPA1 activity reduces melanin synthesis in human epidermal melanocytes; however, the mechanism remains unknown. This study aimed to investigate the roles and mechanism(s) of action of TRPA1 in keratinocytes. The correlation between TRPA1 expression levels and the ability of keratinocytes to phagocytize melanosomes was examined using melanin silver staining. TRPA1 depleted human epidermal keratinocytes and keratinocyte cell lines HaCaT were established using adenovirus-expressing shRNAs against TRPA1. The effects of TRPA1 on keratinocytes and HaCaT cells were determined using cell-based analyses, including light stimulation, calcium imaging, melanin phagocytosis, immunoblotting, and co-immunoprecipitation assays. The degree of epidermal pigmentation was determined in a guinea pig model. TRPA1 mediated the phagocytic activity of keratinocytes. TRPA1 knockdown markedly suppressed melanosome transport to keratinocytes. Mechanistically, TRPA1 was required for PAR-2-induced melanosome phagocytosis in keratinocytes. Furthermore, TRPA1 activation indirectly stabilized microtubules by promoting the competitive binding of CYLD and acetylated α-tubulin. In addition, bortezomib (PS-341), a proteasome inhibitor, increased TRPA1 and CYLD expression and promoted phagocytic activity both in vitro and in vivo. Our findings firstly suggest that TRPA1 promotes melanosome transport in keratinocytes and reveal that TRPA1 is a regulator of PAR-2 activation and microtubule stability via the PAR-2/CYLD axis. • TRPA1 mediated the phagocytic activity of keratinocytes. • TRPA1 knockdown markedly suppressed melanosome transport to keratinocytes. • TRPA1 activation indirectly stabilized microtubules via competitive binding. • Topical application of PS-341 causes pigmentation effects in vivo. [ABSTRACT FROM AUTHOR]

Colour patterning in extant animals can be used as a reliable indicator of their biology and, in extant fish, can inform on feeding strategy. Fossil fish with preserved colour patterns may thus illuminate the evolution of fish behaviour and community structure, but are understudied. Here we report preserved melanin‐based integumentary colour patterning and internal anatomy of the fossil moonfish Mene rhombea (Menidae) from the Bolca Lagerstätte (Eocene (Ypresian), north‐east Italy). The melanosome‐based longitudinal stripes of M. rhombea differ from the dorsal rows of black spots in its extant relative M. maculata, suggesting that the ecology of moonfish has changed during the Cenozoic. Extant moonfish are coastal schooling fish that feed on benthic invertebrates, but the longitudinal stripes and stomach contents with fish remains in M. rhombea suggest unstructured open marine ecologies and a piscivorous diet. The localized distribution of extant moonfish species in the Indo‐Pacific Ocean may reflect, at least in part, tectonically‐driven reorganization of global oceanographic patterns during the Cenozoic. It is likely that shifts in habitat and colour patterning genes promoted colour pattern evolution in the menid lineage. [ABSTRACT FROM AUTHOR]

Melanophilin (MLPH) functions as a linker between RAB27A and myosin Va (MYO5A) in regulating skin pigmentation during the melanosome transport process. The MYO5A-MLPH-RAB27A ternary protein complex is required for anchoring mature melanosomes in the peripheral actin filaments of melanocytes for subsequent transfer to adjacent keratinocytes. Griscelli syndrome type 3 (GS3) is caused by mutations in the MLPH gene. So far, only five variants of MLPH associated with GS3 have been reported. Here, we reported the first patient with GS3 in a Chinese population. The proband carried a novel homozygous missense mutation (c.73G>C; p.D25H), residing in the conserved Slp homology domain of MLPH, and presented with hypopigmentation of the hair, eyebrows, and eyelashes. Light microscopy revealed the presence of abnormal pigment clumping in his hair shaft. In silico tools predicted this MLPH variant to be likely pathogenic. Using immunoblotting and immunofluorescence analysis, we demonstrated that the MLPH (D25H) variant had an inhibitory effect on melanosome transport by exhibiting perinuclear melanosome aggregation in melanocytes, and greatly reduced its binding to RAB27A, although the protein level of MLPH in the patient was not changed. Our findings suggest that MLPH (D25H) is a pathogenic variant that expands the genetic spectrum of the MLPH gene.

Molecular motors of the kinesin superfamily (KIF) are a class of ATP-dependent motor proteins that transport cargo, including vesicles, along the tracks of the microtubule network. Around 45 KIF proteins have been described and are grouped into 14 subfamilies based on the sequence homology and domain organization. These motors facilitate a plethora of cellular functions such as vesicle transport, cell division and reorganization of the microtubule cytoskeleton. Current studies suggest that KIF13A, a kinesin-3 family member, associates with recycling endosomes and regulates their membrane dynamics (length and number). KIF13A has been implicated in several processes in many cell types, including cargo transport, recycling endosomal tubule biogenesis, cell polarity, migration and cytokinesis. Here we describe the recent advances in understanding the regulatory aspects of KIF13A motor in controlling the endosomal dynamics in addition to its structure, mechanism of its association to the membranes, regulators of motor activity, cell type-specific cargo/membrane transport, methods to measure its activity and its association with disease. Thus, this review article will provide our current understanding of the cell biological roles of KIF13A in regulating endosomal membrane remodeling.

GPCRs transform extracellular stimuli into a physiological response by activating an intracellular signaling cascade initiated via binding to G proteins. Orphan G protein-coupled receptors (GPCRs) hold the potential to pave the way for development of new, innovative therapeutic strategies. In this review we will introduce G protein-coupled receptor 143 (GPR143), an enigmatic receptor in terms of classification within the GPCR superfamily and localization. GPR143 has not been assigned to any of the GPCR families due to the lack of common structural motifs. Hence we will describe the most important motifs of classes A and B and compare them to the protein sequence of GPR143. While a precise function for the receptor has yet to be determined, the protein is expressed abundantly in pigment producing cells. Many GPR143 mutations cause X-linked Ocular Albinism Type 1 (OA1, Nettleship-Falls OA), which results in hypopigmentation of the eyes and loss of visual acuity due to disrupted visual system development and function. In pigment cells of the skin, loss of functional GPR143 results in abnormally large melanosomes (organelles in which pigment is produced). Studies have shown that the receptor is localized internally, including at the melanosomal membrane, where it may function to regulate melanosome size and/or facilitate protein trafficking to the melanosome through the endolysosomal system. Numerous additional roles have been proposed for GPR143 in determining cancer predisposition, regulation of blood pressure, development of macular degeneration and signaling in the brain, which we will briefly describe as well as potential ligands that have been identified. Furthermore, GPR143 is a promiscuous receptor that has been shown to interact with multiple other melanosomal proteins and GPCRs, which strongly suggests that this orphan receptor is likely involved in many different physiological actions.

Dysregulation of melanin homeostasis is implicated in causing skin pigmentation disorders, such as melasma due to hyperpigmentation and vitiligo due to hypopigmentation. Although the synthesis of melanin has been well studied, the removal of the formed skin pigment requires more research. We determined that β-mangostin, a plant-derived metabolite, induces the degradation of already-formed melanin in the mouse B16F10 cell line. The whitening effect of β-mangostin is mediated by macroautophagy/autophagy, as it was abolished by the knockdown of ATG5 or RB1CC1/FIP200, and by treatment with 3-methyladenine, a phosphatidylinositol 3-kinase complex inhibitor. However, the exact autophagy mechanism of melanosome degradation remains unknown. Selective autophagy for a specific cellular organelle requires specific E3-ligases and autophagic receptors for the target organelle. In this study, an E3-ligase, RCHY1, and an autophagy receptor, OPTN (optineurin), were identified as being essential for melanophagy in the β-mangostin-treated B16F10 cell line. As per our knowledge, this is the first report of a specific mechanism for the degradation of melanosomes, the target organelle of melanophagy. These findings are expected to broaden the scope of melanin homeostasis research and can be exploited for the development of therapeutics for skin pigmentation disorders. [ABSTRACT FROM AUTHOR]

During the laser treatment of pigmented dermatosis such as Nevus of Ota, vapor bubbles will be generated by the laser with short pulse width and high energy density. Laser irradiation is efficacious for the clinical treatment of Ota's Nevus caused by hyperplasia of melanosomes in dermis. Since the mechanism of the laser–melanosome interaction is not yet clear, the clearance rate is generally low and bleeding of irradiated skin frequently occurs. This work conducted a flow visualization experiment to investigate the laser–melanosome interaction mechanism by using high-speed imaging. Pigmented phantom was prepared to simulate the diseased dermis tissue, where agar acted as substrate and synthetic melanin particles was infused as hyperplastic melanosomes. Putting the phantom into water, its thermo-mechanical responses to single-pulse 1064-nm Nd:YAG laser irradiation with energy density of 4–7 J/cm2 and pulse duration of 6 ns were recorded. The results indicated that laser-induced bubble formation caused by the gasification of tissue moisture is the key mechanism of laser–melanosome interaction, and an optimal energy density of 6 J/cm2 is recommended. [ABSTRACT FROM AUTHOR]

Hair contains about 80% keratins and 1–3% melanin packaged in melanosomes. Both of these are high-value and functional raw materials that have potential applications in wide-ranging fields. While keratin extraction has been widely refined, efficient methods of melanosome extraction are limited. The extraction of melanosomes requires complete removal of keratin, thus combining keratin extraction and melanosome isolation is logical. Herein, a successive process to harvest melanosomes after keratin extraction from human hair waste was developed. The yield of melanosomes was about 1.3% of the total hair mass. The structure of harvested melanosomes is well preserved based on surface morphology and interior ultrastructural observations using electron microscopy. The chemical structure, ultraviolet (UV)-filtering ability, and thermal stability of the melanosomes are examined to demonstrate preservation of native functions. Our strategy of combining melanosome isolation with keratin extraction is shown to be effective and significantly improves the total resource recovery efficiency from human hair waste. Effective harvesting of functional melanosomes following keratin extraction from human hair. [ABSTRACT FROM AUTHOR]

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 < 0.001). Light‐skinned subjects had less HRD than dark‐skinned subjects. Conclusion: We observed light‐induced modifications in the space between the EZ and the IZ. We hypothesize that the HRDs visible in this zone correspond to melanosomes that are mobilized during the light stimulation protocol. Larger studies are recommended to further evaluate and confirm light‐induced SD‐OCT changes under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Marine deposits of earliest Eocene age in northern Jutland, Denmark, are renowned for yielding diverse teleost assemblages that have proved central for enhancing our understanding of the early evolution of many extant actinopterygian clades. In this study, we investigate diminutive larval fish fossils from the Stolleklint Clay, Ølst Formation, that retain multiple soft‐tissue features preserved as distinct dark‐coloured stains. To examine the elemental and molecular composition of these soft parts, we employed a combination of time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), scanning electron microscopy (SEM) and energy‐dispersive x‐ray spectroscopy (EDS). Our analyses revealed that the preserved structures contain chemically identifiable eumelanin intimately associated with densely aggregated microbodies that are morphologically consistent with melanosome organelles. Thus, we conclude that the carbonaceous structures represent traces of originally melanized body parts, including the eyes and peritoneum. Comparable pigmentation patterns are seen in many extant teleost larvae that use semi‐transparency as a means of camouflage in pelagic environments, to suggest a similar visual appearance of the Stolleklint Clay fish fossils. This in turn suggests that adaptations for concealment and UV‐protection had already evolved by the beginning of the Eocene, notably during a time interval characterized by an extreme greenhouse climate, when the global fish fauna become increasingly modern in composition. [ABSTRACT FROM AUTHOR]

Oculocutaneous albinism (OCA) is a heritable disorder of pigment production that manifests as hypopigmentation and altered eye development. Exon sequencing of known OCA genes is unsuccessful in producing a complete molecular diagnosis for a significant number of affected individuals. We sequenced the DNA of individuals with OCA using short‐read custom capture sequencing that targeted coding, intronic, and noncoding regulatory regions of known OCA genes, and genome‐wide association study‐associated pigmentation loci. We identified an OCA2 complex structural variant (CxSV), defined by a 143 kb inverted segment reintroduced in intron 1, upstream of the native location. The corresponding CxSV junctions were observed in 11/390 probands screened. The 143 kb CxSV presents in one family as a copy number variant duplication for the 143 kb region. In the remaining 10/11 families, the 143 kb CxSV acquired an additional 184 kb deletion across the same region, restoring exons 3–19 of OCA2 to a copy‐number neutral state. Allele‐associated haplotype analysis found rare SNVs rs374519281 and rs139696407 are linked with the 143 kb CxSV in both OCA2 alleles. For individuals in which customary molecular evaluation does not reveal a biallelic OCA diagnosis, we recommend preliminary screening for these haplotype‐associated rare variants, followed by junction‐specific validation for the OCA2 143 kb CxSV. [ABSTRACT FROM AUTHOR]

B16-F1 melanoma cells have often been used as a model to investigate melanogenesis, but the evidence that melanosome biogenesis and transport occur by the same mechanisms in normal melanocytes and B16-F1 cells is insufficient. In this study, we established knockout B16-F1 cells for each of several key factors in melanogenesis, i.e., tyrosinase (Tyr), Hps4, Rab27A, and Rab32·Rab38 (Rab32/38), and then compared their phenotypes with the phenotypes of corresponding mutant mouse melanocyte cell lines, i.e., melan-c, melan-le, melan-ash, and Rab32-deficient melan-cht cells, respectively. The results showed that Tyr and Rab27A are also indispensable for melanin synthesis and peripheral melanosome distribution, respectively, in B16-F1 cells, but that Hps4 or its downstream targets Rab32/38 are not essential for Tyr transport in B16-F1 cells, suggesting the existence of a Rab32/38-independent Tyr transport mechanism in B16-F1 cells. We then performed comprehensive knockdown screening of Rab small GTPases and identified Rab10 and Rab24, previously uncharacterized Rabs in melanocytes, as being involved in Tyr transport under Rab32/38-null conditions. Our findings indicate a difference between the Tyr transport mechanism in melanocytes and B16-F1 cells in terms of Rab32/38-dependency and a limitation in regard to using melanoma cells as a model for melanocytes, especially when investigating the mechanism of endosomal Tyr transport.

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.

• Melanosomes are concentrated in the basal keratinocytes for localized protection. • Regional distribution of melanosomes correlates with their selective transfer. • Regional distribution of melanosomes correlates with their differential degradation. 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. [ABSTRACT FROM AUTHOR]

Iridescence is widespread in the living world, occurring in organisms as diverse as bacteria, plants, and animals. Yet, compared to pigment-based forms of coloration, we know surprisingly little about the developmental and molecular bases of the structural colors that give rise to iridescence. Birds display a rich diversity of iridescent structural colors that are produced in feathers by the arrangement of melanin-containing organelles called melanosomes into nanoscale configurations, but how these often unusually shaped melanosomes form, or how they are arranged into highly organized nanostructures, remains largely unknown. Here, we use functional genomics to explore the developmental basis of iridescent plumage using superb starlings (Lamprotornis superbus), which produce both iridescent blue and non-iridescent red feathers. Through morphological and chemical analyses, we confirm that hollow, flattened melanosomes in iridescent feathers are eumelanin-based, whereas melanosomes in non-iridescent feathers are solid and amorphous, suggesting that high pheomelanin content underlies red coloration. Intriguingly, the nanoscale arrangement of melanosomes within the barbules was surprisingly similar between feather types. After creating a new genome assembly, we use transcriptomics to show that non-iridescent feather development is associated with genes related to pigmentation, metabolism, and mitochondrial function, suggesting non-iridescent feathers are more energetically expensive to produce than iridescent feathers. However, iridescent feather development is associated with genes related to structural and cellular organization, suggesting that, while nanostructures themselves may passively assemble, barbules and melanosomes may require active organization to give them their shape. Together, our analyses suggest that iridescent feathers form through a combination of passive self-assembly and active processes. [ABSTRACT FROM AUTHOR]

Summary: The cyclic AMP pathway promotes melanocyte differentiation by activating CREB and the cAMP-regulated transcription co-activators 1–3 (CRTC1–3). Differentiation is dysregulated in melanomas, although the contributions of CRTC proteins is unclear. We report a selective differentiation impairment in CRTC3 KO melanocytes and melanoma cells, due to downregulation of oculo-cutaneous albinism II (OCA2) and block of melanosome maturation. CRTC3 stimulates OCA2 expression by binding to CREB on a conserved enhancer, a regulatory site for pigmentation and melanoma risk. CRTC3 is uniquely activated by ERK1/2-mediated phosphorylation at Ser391 and by low levels of cAMP. Phosphorylation at Ser391 is constitutively elevated in human melanoma cells with hyperactivated ERK1/2 signaling; knockout of CRTC3 in this setting impairs anchorage-independent growth, migration, and invasiveness, whereas CRTC3 overexpression supports cell survival in response to the mitogen-activated protein kinase (MAPK) inhibitor vemurafenib. As melanomas expressing gain-of-function mutations in CRTC3 are associated with reduced survival, our results suggest that CRTC3 inhibition may provide therapeutic benefit in this setting.

Tyrosinase (TYR) is a copper-containing monooxygenase central to the function of melanocytes. Alterations in its expression or activity contribute to variations in skin, hair and eye color, and underlie a variety of pathogenic pigmentary phenotypes, including several forms of oculocutaneous albinism (OCA). Many of these phenotypes are linked to individual missense mutations causing single nucleotide variants and polymorphisms (SNVs) in TYR. We previously showed that two TYR homologues, TYRP1 and TYRP2, modulate TYR activity and stabilize the TYR protein. Accordingly, to investigate whether TYR, TYRP1, and TYRP2 are biophysically compatible with various heterocomplexes, we computationally docked a high-quality 3D model of TYR to the crystal structure of TYRP1 and to a high-quality 3D model of TYRP2. Remarkably, the resulting TYR-TYRP1 heterodimer was complementary in structure and energy with the TYR-TYRP2 heterodimer, with TYRP1 and TYRP2 docking to different adjacent surfaces on TYR that apposed a third realistic protein interface between TYRP1-TYRP2. Hence, the 3D models are compatible with a heterotrimeric TYR-TYRP1-TYRP2 complex. In addition, this heterotrimeric TYR-TYRP1-TYRP2 positioned the C-terminus of each folded enzymatic domain in an ideal position to allow their C-terminal transmembrane helices to form a putative membrane embedded three-helix bundle. Finally, pathogenic TYR mutations causing OCA1A, which also destabilize TYR biochemically, cluster on an unoccupied protein interface at the periphery of the heterotrimeric complex, suggesting that this may be a docking site for OCA2, an anion channel. Pathogenic OCA2 mutations result in similar phenotypes to those produced by OCA1A TYR mutations. While this complex may be difficult to detect in vitro, due to the complex environment of the vertebrate cellular membranous system, our results support the existence of a heterotrimeric complex in melanogenesis.