Melanosome Formation Research Articles

Identification of a keratin-associated protein with a putative role in vesicle transport

Protection of skin against UV light requires a coordinated interaction between melanocytes and keratinocytes. Melanosomes are lysosome-related organelles that originate in melanocytes and are transferred into keratinocytes where they form a supranuclear cap. The mechanism responsible for melanosome transfer into keratinocytes and their intracellular distribution is poorly understood. Recently, we reported for the first time that loss-of-function mutations in the keratin K5 gene affect melanosome distribution in keratinocytes and results in a reticulate hyperpigmentation disorder, called Dowling–Degos disease. Here, we characterise the distribution and behaviour of individual K5 and K14 domains following transient and stable transfection into cells. We report that the K5 head domain is considerably more stable than the K14 head. Moreover, the distribution of the K5 head domain is altered following depolymerisation of microtubules. Following co-immunoprecipitation, we verified a specific interaction between the head domain of K5 with Hsc70, a chaperone also involved in vesicle uncoating. We hypothesise that this interaction is involved in melanosome formation or transport in keratinocytes. Alternatively, it may have a general function in the regulation of keratin assembly.

The zebrafish mutant vps18 as a model for vesicle‐traffic related hypopigmentation diseases

Hypopigmentation is a characteristic of several diseases associated with vesicle traffic defects, like the Hermansky-Pudlak, Chediak-Higashi, and Griscelli syndromes. Hypopigmentation is also a characteristic of the zebrafish mutant vps18(hi2499A), which is affected in the gene vps18, a component of the homotypic fusion and protein sorting complex that is involved in tethering during vesicular traffic. Vps18, as part of this complex, participates in the formation of early endosomes, late endosomes, and lysosomes. Here, we show that Vps18 is also involved in the formation of melanosomes. In the zebrafish mutant vps18(hi2499A) the retroviral insertion located at exon 4 of vps18, leads to the formation of two abnormal splicing variants lacking the coding sequence for the clathrin repeat and the RING finger conserved domains. A deficiency of Vps18 in zebrafish larvae results in hepatomegaly and skin hypopigmentation. We also observed a drastic reduction in the number of melanosomes in the eye's retinal pigmented epithelium along with the accumulation of immature melanosomes. A significant reduction in the vps18(hi2499A) larvae visual system capacity was found using the optokinetic response assay. We propose that the insertional mutant vps18(hi2499A) can be used as a model for studying hypopigmentation diseases in which vesicle traffic problems exist.

Association of the Hermansky-Pudlak syndrome type-3 protein with clathrin

BackgroundHermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172–176.ResultsClathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20°C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution.ConclusionHPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking.

Genetics of pigmentary disorders.

The genetic and molecular bases of various types of congenital pigmentary disorders have been classified in the past 10 years, as follows: (1) disorders of melanoblast migration in the embryo from the neural crest to the skin: piebaldism; Waardenburg syndrome 1-4 (WS1-WS4); dyschromatosis symmetrica hereditaria. (2) Disorders of melanosome formation in the melanocyte: Hermansky-Pudlak syndrome 1-7 (HPS1-7); Chediak-Higashi syndrome 1 (CHS1). (3) Disorders of melanin synthesis in the melanosome: oculocutaneous albinism 1-4 (OCA1-4). (4) Disorders of mature melanosome transfer to the tips of the dendrites Griscelli syndrome 1-3 (GS1-3). These disorders are presented and their gene mutations and pathogenesis are discussed.

Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma.

Protoporphyrin IX (PpIX) synthesis by malignant cells is clinically exploited for photodiagnosis and photodynamic therapy following administration of 5-aminolevulinic acid (ALA). The expression and activity of the housekeeping porphobilinogen deaminase (PBGD) was correlated to PpIX synthesis in differentiating B16 melanoma cells. Differentiation was stimulated by two inducers, butyrate and hexamethylene bisacetamide (HMBA), both of which promote the formation of typical melanosomes and melanin, as well as morphological changeover. A marked decrease in total PBGD activity and PpIX synthesis was observed following stimulation by butyrate, while HMBA induced an opposite effect. In contrast, ferrochelatase levels remained unchanged. Photodynamic inactivation of the cells undergoing differentiation was largely dependent on the PpIX accumulation, which was modulated by the two inducers butyrate and HMBA. Fluorescence immunostaining with anti-PBGD antibodies revealed a major PBGD fraction in the nucleus and a minor fraction in the cytosol. This nuclear localisation pattern was confirmed by expression of PBGD fused to green fluorescence protein. We suggest that efficient photodynamic therapy of cancer facilitated by ALA administration can be enhanced using combined therapeutic modalities.

Transplantable melanomas in gerbils (Meriones unguiculatus). II: melanogenesis.

We characterized the melanogenic apparatus in a family of transplantable gerbil melanomas (melanotic and amelanotic) using a combination of biophysical, ultrastructural and biochemical methods. Melanotic melanomas produced pure eumelanin but in vesiculo-globular melanosomes ('pheomelanosomes'); the eumelanosomes, characteristically ellipsoidal in shape with fibrillar or fibrillo-lamelar matrix, were never noticed. Melanotic melanomas also had significant tyrosinase activity and Zn, Pb/S, Ca and P content; all higher than in the amelanotic variants. The amelanotic variant, which was devoid of melanin pigment and melanosomes, had clearly detectable tyrosinase activity (albeit at 20% of that in the melanotic variant). Thus, with these multidirectional approaches we demonstrate that pure eumelanin can be synthesized in organelles ultrastructurally defined as pheomelanosomes, but a defect in the formation of melanosomes can prevent in vivo melanin synthesis despite the presence of detectable tyrosinase activity. We conclude that this melanoma system provides an excellent experimental model for the study of molecular components determining pheo- and/or eumelanogenesis. The information generated can be used for defining the roles of melanogenesis and of tyrosinase expression in the regulation of melanoma behavior and the effect of their modification on the course of the disease.

Melanosomes and MHC class II antigen-processing compartments: a tinted view of intracellular trafficking and immunity.

Melanosomes are specialized intracellular compartments within melanocytes and retinal pigment epithelial cells that function in the synthesis, storage, and secretion of melanins, which are the major pigments made by mammals. The mechanisms that regulate the formation of melanosomes, and the pathways by which constituent proteins are targeted to them, are related to those involved in the biogenesis of major histocompatibility complex (MHC) class II antigen-processing compartments. Consequently, diseases that affect pigmentation may also affect antigen presentation to T cells. Moreover, many of the tissue-specific proteins that localize to melanosomes and participate in melanin formation double as tumor-associated antigens that are targets for T cells in patients with melanoma. Our studies on melanosome biogenesis are providing new ways of thinking about antigen-processing compartments and the mechanisms regulating presentation of tumor-associated antigens.

Genetic and Epigenetic Control of the Proliferation and Differentiation of Mouse Epidermal Melanocytes in Culture

Serum-free culture of epidermal cell suspensions from neonatal skin of mice of strain C57BL/10JHir (B10) showed that alpha-melanocyte-stimulating hormone (alpha-MSH) was involved in regulating the differentiation of melanocytes by inducing tyrosinase activity, melanosome formation, and dendritogenesis. Dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) similarly induced the differentiation of melanocytes. On the other hand, DBcAMP induced the proliferation of epidermal melanocytes in culture in the presence of keratinocytes. Basic fibroblast growth factor (bFGF) was also shown to stimulate the sustained proliferation of undifferentiated melanoblasts in the presence of DBcAMP and keratinocytes. These results suggest that the proliferation and differentiation of mouse epidermal melanoblasts and melanocytes in culture are regulated by the three factors; namely, cAMP, bFGF, and keratinocyte-derived factors. Moreover, serum-free primary culture of mouse epidermal melanocytes derived from B10 congenic mice, which carry various coat color genes, showed that the coat color genes were involved in regulating the proliferation and differentiation of mouse epidermal melanocytes by controlling the proliferative rate, melanosome formation and maturation, and melanosome distribution.

Pharmacological Disruption of Hair Follicle Pigmentation by Cyclophosphamide as a Model for Studying the Melanocyte Response to and Recovery from Cytotoxic Drug Damage In Situ

Here we show that cyclophosphamide induces disruption of follicular melanogenesis, which is characterized by abnormal transfer of pigment granules to ectopic hair bulb locations, extrafollicular melanin incontinence, disordered formation of melanosomes, and inhibition of melanosome transfer into precortical keratinocytes. This is in contrast to dexamethasone-induced termination of follicle melanogenesis, which activates premature but predominantly normal catagen development. Cyclophosphamide-induced pigmentation disruption was accompanied by significant alterations of biochemical and biophysical markers of melanogenesis, compared to control mice treated either with vehicle or with topical dexamethasone. Electron paramagnetic resonance spectroscopy shows a decline in the melanin signal and predominant eumelanin production. Tyrosine hydroxylase activity of tyrosinase and dihydroxyphenylalanine oxidation drop rapidly, while DOPAchrome tautomerase activity increases and dihydroxyindole carboxylic acid conversion factor activity remains unchanged in cyclophosphamide-treated mice compared to controls. These observations emphasize the key role of tyrosinase as opposed to postdihydroxyphenylalanine oxidase steps in normal and pathological termination of melanogenesis and shows that tyrosinase is the most sensitive target of the melanogenic apparatus for pharmacological regulation. Follicle pigmentation recovers only during the subsequent hair cycle, i.e., after a new anagen hair bulb has been constructed, which points to the existence of a relatively chemoresistant melanoblast-like cell population residing in the noncycling part of the hair follicle.

Melanosome Formation in Cultured Amelanotic Melanophores of Rana brevipoda by a Frog Tyrosinase cDNA Transfection

A cDNA encoding tyrosinase of Rana nigromaculata was introduced into cultured, tyrosinase-negative amelanotic melanophores of R. brevipoda by a calcium phosphate precipitation method. Within a few days following transfection, dark pigmentation became visible in a small number of cells. Light microscopic observation revealed that the morphology of these transformed cells was comparable to that of normal melanophores in culture, and their proliferative activity was lower than that of amelanotic cells. Ultrastructural examination verified that amelanotic melanophores possessed a relatively small number of premelanosomes while the transformants contained numerous melanosomes at various stages of pigment deposition. The result indicated that tyrosinase cDNA of R. nigromaculata was expressed in amelanotic melanophores of R. brevipoda including the maturation of premelanosomes. It was also suggested that the expression of transfected tyrosinase cDNA had promoted differentiation of the amelanotic cells into fully developed melanophores.

UV-Irradiated Melanocytic Nevi Simulating Melanoma In Situ

A causative role of UV light in the development of melanocytic neoplasms has often been suggested. In order to investigate the short-term effects of UV light on melanocytic nevi, the morphological and immunohistochemical changes in nevi after a single UV irradiation are studied in 12 nevi from 10 patients and compared with the nonirradiated part of the same nevus. After irradiation more melanocytes above the dermal-epidermal junction are observed in seven nevi, simulating a melanoma in situ in three nevi. Moreover, a marked increase in the expression of HMB-45 is found after irradiation in all investigated nevi, indicating an activation of the melanocytes and active melanosome formation. The metabolic activity correlates with the ultrastructural findings, which show a large cytoplasm, hypertrophic Golgi apparatus, abundant mitochondria, and an increased number of melanosomes of different stages. One week after irradiation, no increase in the proliferative activity of the melanocytes is found. The morphological and immunohistochemical changes after one low dose of UV irradiation should be considered in the differential diagnosis of pigmented skin lesions. The UV-irradiated nevus should be added to the list of so-called simulators of malignant melanoma.

Formation of pterinosomes and carotenoid granules in xanthophores of the teleost Oryzias latipes as revealed by the rapid-freezing and freeze-substitution method

The rapid-freezing and freeze-substitution method was applied for the ultrastructural study of the dermal chromatophores of a teleost, Oryzias latipes. The method was found to be suitable for preserving fragile membranous structures within melanophores and xanthophores. In addition, relatively high electron density in overall profile indicates that the procedure is effective in reducing the extraction of cytoplasmic ground substances that inevitably occurs during the process of conventional chemical fixation and the following dehydration. The improved ultrastructural images clearly show that the pterinosomes, the characteristic pigmentary organelles of xanthophores, are formed through several distinct developmental stages starting from the loose congregations of vesicles derived from the Golgi complex. The earlier stages of development are similar to those found in melanosome formation. Whereas carotenoid pigments in xanthophores in conventional aldehyde-osmium-fixed materials are found to be electrondense membrane-free particles, they are identified as membrane-bounded organelles in the present study. The envelope of these carotenoid vesicles does not exhibit a typical trilaminar structure but appears to be an extremely thin membrane. Carotenoid vesicles are, in most cases, in direct contact with the outer surface of tubular endoplasmic reticulum.

Control of melanocyte proliferation and differentiation in the mouse epidermis.

Melanocyte-stimulating hormone plays an important role in the regulation of melanocyte differentiation in the mouse epidermis by inducing tyrosinase activity, melanosome formation, translocation of melanosomes, and increased dendritogenesis. The proliferative activity of differentiating epidermal melanocytes of newborn mice during the healing of skin wounds is regulated by semi-dominant genes, suggesting that the genes are involved in regulating the proliferative activity of epidermal melanocytes during differentiation. From the results of serum-free culture of epidermal cell suspensions from neonatal mouse skin, basic fibroblast growth factor is shown to stimulate the sustained proliferation of melanoblasts in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate and keratinocyte-derived factors. Moreover, each step of melanocyte differentiation is controlled by numerous coat color genes. These genes control melanocyte differentiation by regulating the differentiation of neural crest cells into melanoblasts in embryonic skin, or by regulating the differentiation of neural crest cells into melanoblasts in embryonic skin, or by regulating transcription and/or translation of the tyrosinase gene in the differentiating melanocytes. These results suggest that melanocyte proliferation and differentiation in the mouse epidermis are controlled by both genetic factors and local tissue environment.

Cell adhesion regulates melanoma specific differentiation and interactions with the 3′ region of the tyrosinase gene

Decreased attachment to substratum has now been found to increase melanosome formation and cell-cell interaction in B16 melanoma. Since melanosome formation involves tyrosinase gene expression, we assayed for differential RNA expression by hybridization with probes from the distal ends of this gene, detecting unequal reactivity only with the 3′ end probe. The same DNA showed binding of 2 nuclear proteins of 50 and 60 kd in unanchored cells, in contrast with a decreased binding of the 60 kd species, in nuclear extracts from attached cells. No comparable differences were detected with a γ-actin DNA of identical length, suggesting that the changes observed are sequence-specific. Our studies suggest that the adhesion-mediated modulation of pigmentation in B16 melanoma correlates with differential macromolecular interactions with the 3′ end of the tyrosinase gene.

Reversible dedifferentiation and redifferentiation of a melanized cell line from a goldfish tumor

We reported previously the isolation of a melanized cell line that can undergo reversible dedifferentiation and redifferentiation. A heavily pigmented cell line, designated as P15, originally isolated by fish serum-induced melanization of some GEM 81 cells, cloned and serially passaged in fish serum medium, became noticeably less pigmented after several months in fetal calf serum medium and completely unpigmented after another year in the same medium. Addition of fish serum to the medium of this dedifferentiated cell line, designated P15D, induced pigmentation within a week. This re-induced pigmented cell line, designated as P15DI, became unpigmented when cultured in fetal calf serum medium for one month. We report here that the dedifferentiation of P15 occurs in two stages. One week after withdrawal of fish serum, the specific activity of tyrosinase of the culture dropped by ∼ 70% and remained at this reduced level for at least one month. After one year, the specific activity of tyrosinase had dropped to a barely detectable level and the culture became completely unpigmented (P15D). Electron microscopic studies showed that the P15D cells have no melanosomes, probably no large vesicles for melanosome formation, but some dopa-positive trans-Golgi network (TGN). Addition of fish serum to the growth medium of P15 cultures led to a steady increase in the specific activity of tyrosinase, detectable after one day. There was also an increase in the amount of dopa-positive TGN within one day. Melanosomes first appeared after three days and became numerous after one week. Upon removal of fish serum, these re-induced cells (P15DI) underwent a rapid decrease in the specific activity of tyrosinase, reaching, after eight days, the basal level seen in P15D cells. We also report that a protein designated as p75 ( M r ∼ 75 000), previously shown to be associated with melanosomes in two melanized cell types of goldfish origin, is present in all melanized cell lines, including P15 and P15DI but absent in unmelanized cell lines, including P15D.

Hypopigmentation in dogs treated with an inhibitor of platelet aggregation.

Hypopigmentation was the principal manifestation of systemic toxicity when the experimental platelet aggregation inhibitor, PD-89454, was orally administered to Beagle dogs for 28 days. Pigmentation changes were present in skin of nose, lips and eyelids and mucosa of hard palate. Fontana-Masson stain demonstrated decreased melanin within melanocytes and keratinocytes. Melanocytes were globoid with inconspicuous dendritic processes and had small, incompletely pigmented melanosomes that were quantitatively reduced relative to controls. Toxic mechanism of action of PD-89454, structurally distinct from known depigmenting compounds, was not established but ultrastructural findings suggest an interference in melanosome formation and/or melanization.

Spreading Pigmented Actinic Keratosis

Spreading pigmented actinic keratosis is a rarely reported premalignant, pigmented, epidermal tumor that can be difficult to distinguish clinically from other benign and malignant pigmented lesions. In an attempt to determine the cause of clinical hyperpigmentation, seven lesions from five patients were examined by light microscopy. Three of these lesions were also examined using electron microscopy. Findings from these studies indicate that hyperpigmentation is due to enhanced melanosome formation and distribution and not to a melanocyte-keratinocyte melanosome transfer block.

On the formation and degradation of melanosomes in smooth muscle cells: electron microscopic investigation on the m. sphincter pupillae of the rat.

The electron microscopic investigation of the m. sphincter pupillae of adult black hooded rats showed the presence of melanosomes in the smooth muscle cells. In shape and size the melanosomes were like those of the iridial epithelium. In addition, premelanosomes and tyrosinase activity were observed as well as melanosomes with disintegrated content and acid phosphatase activity. The data suggest that the smooth muscle cells of the m. sphincter pupillae are capable of the formation and degradation of melanosomes.

Deficient melanosome formation in some coat-color mutant mice revealed by a monoclonal antibody against melanosome.

Some coat-color loci in mice are considered to control melanosome formation. In order to investigate genetic control of melanosome-associated proteins, we prepared monoclonal antibodies against mouse melanosomes. Melanosomes were isolated from B16 mouse melanoma through differential fractionation. BALB/c mice were immunized with an SDS-solubilized melanosome fraction. The spleen cells were subsequently fused with mouse myeloma cells, the resulting hybridomas cloned. Their secreted IgG was screened for reactivity to the SDS-solubilized melanosome fraction. One monoclonal antibody, M10, was shown to react to melanosomes by immunoelectronmicroscopy. It recognized a single protein band of 61,000 dalton on immunoblots of gel-fractionated melanosomes. The reactivities of M10 to skin homogenates from various coat-color mutants were examined by the ELISA method. Five congenic genotypes, non-agouti (a/a), brown (b/b), albino (c/c), dilute (d/d), and pink-eyed dilution (p/p) were examined. Among these, b/b and p/p showed significantly lower reactivities than a/a. Our results seem to suggest that the pigment abnormalities in these mutants result from abnormalities of the melanosomal proteins. In the case of albino mice, the reactivity of M10 to skin homogenate was almost the same as the wild-type mouse. It seems that the albino mice are capable of producing the melanosomal protein.

Growth and maturation of melanosomes in the melanophores of a teleost, Oryzias latipes

Formation of melanosomes in melanophores of a teleost, Oryzias latipes, was studied by means of electron microscopy. Two distinct types of premelanosomes are observed in the same cell: (i) multivesicular premelanosomes, which later develop into melanosomes with electron-lucent “hollows” in the center, appear at early embryonic stages; (ii) premelanosomes with highly organized, fibrous internal structure are formed at later stages of development and give rise to melanosomes with a filamentous center. Melanosomes are generally ellipsoid in shape, and the difference in the dimensions of fibrillar premelanosomes, melanosomes in the cells at younger developmental stages and those developed fully in melanophores of adults indicates that these organelles grow during development. The growth is achieved by fusion of small unmelanized vesicles or fibrillar premelanosomes to preformed melanosome and by fusion of two or more premelanosomes to form a larger organelle. The addition of the matrix of fibrillar premelanosomes around preformed melanosomes, which are derived from either multivesicular or fibrillar premelanosomes, forms a concentric outer deposit, and the fusion of small vesicles produces electron-lucent pits which are scattered irregularly in mature melanosomes.