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At least 19 recordsLinked to original sources
PubMed2001

A model for melanosome biogenesis based on the purification and analysis of early melanosomes.

Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.

Electrophoresis↗
PubMed2001

Melanosomal pH controls rate of melanogenesis, eumelanin/phaeomelanin ratio and melanosome maturation in melanocytes and melanoma cells.

The skin pigment melanin is produced in melanocytes in highly specialized organelles known as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal pathway and can have a low internal pH. In the present study we have shown that melanin synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the role of intramelanosomal pH as a possible control mechanism for melanogenesis. To do this we examined the effect of neutralizing melanosomal pH on tyrosinase activity and melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte cultures (9 of 9) from Caucasian skin as well as two melanoma cell lines with comparable melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy revealed an accumulation of melanin and increased maturation of melanosomes in response to pH neutralization. In summary, our findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is an essential factor which regulates multiple stages of melanin production. Furthermore, since we have recently identified that pink locus product (P protein) mediates neutralization of melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We would further propose that the wide variations in both constitutive and facultative skin pigmentation seen in the human population could be associated with the high degree of P-locus polymorphism.

Anti-Bacterial Agents↗
PubMed1982

Characterization of biochemical properties of melanosomes for structural and functional differentiation: analysis of the compositions of lipids and proteins in melanosomes and their subfractions.

Two types of melanosomes were isolated from B16 and Harding Passey (HP) mouse melanomas to elucidate whether there are any features of melanosomal lipids and proteins related to the differences in morphology of melanosomes and in the biological activities of melanoma cells. Biochemical analyses were made to clarify (a) the lipid components of the 2 melanosomes and (b) the lipid and polypeptide compositions of their subfractions, i.e., the outer surface and inner core, resolved by a detergent, Brij-35, separately. We found (a) that the lipid contents in B16 melanosomes were much higher than those in HP, (b) that the B16 and HP melanosomes could be fractionated into a phospholipid-rich outer surface and phospholipid-poor core, (c) that both outer surface and core subfractions of HP were distinct from the corresponding subfractions of B16 with respect to phospholipid contents, (d) that the outer surface of B16 and HP revealed a polypeptide composition similar to each other, although the protein contents of the outer surface were much lower than those of core and (e) that the total melanosomes showed a marked difference in polypeptides between B16 and HP. In addition, both B16 and HP melanosomes revealed the alteration of lipid compositions, e.g., fatty acid acyl chain, similar to that observed during malignant transformation. There was not, however, any significant difference in acyl compositions between B16 and HP. It is likely that melanosomal lipid and protein not only affect the different morphogenesis of melanosomes but also reflect the different biological activities of whole cells, and that lipids, primarily located in the outer surface, regulate the functional aspects of melanosomes.

Animals↗
PubMed1988

Nature and biosynthesis of structural matrix protein in melanosomes: melanosomal structural protein as differentiation antigen for neoplastic melanocytes.

Two major classes of protein, i.e., structural matrix and enzymic, exist in the melanosome. In normal melanocytes, synthesis of these two components is under a strict genetic control with regulatory steps associated with transcription and translation coded with appropriate pigment genes. In abnormal neoplastic melanocytes, they become markedly aberrant. The aberrant melanogenesis can be typically manifested by an abnormality in fine structure of the melanosome. The fine structural heterogeneity of the melanosome can often be diagnostic to certain forms of malignant melanoma and dysplastic melanocytic nevus. This study brings about the importance of the structural matrix protein for the expression of fine structural heterogeneity in the melanosome by developing the 2 monoclonal antibodies, MoAb HMSA-1 and HMSA-2. The 2 MoAbs recognized the cytoplasmic antigen on paraffin embedded sections, which under immunoelectron microscopy and cell fractionation studies, were found to be localization the melanosome and smooth ER, but not Golgi complex and coated vesicles as seen in the tyrosinase studies. It is indicated (a) that the sites of the synthesis for the melanosomal protein and tyrosinase are different, (b) that the melanosomal structural protein is accumulated in the dilated vacuole of smooth ER from which the stage I melanosome is synthesized, (c) that the synthesis of melanosomal protein becomes markedly aberrant and directly reflects the abnormal fine structure of the melanosome and (d) the heterogeneity in synthesis of melanosomal protein as expressed by MoAb HMSA-1 and HMSA-2 is a useful adjunct for immunohistopathological differentiation of malignant melanoma cells from benign nevoid cells and normal melanocytes.

Antigens, Differentiation↗
PubMed1987

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.

Animals↗
PubMed1978

Uptake of melanosomes and increased melanin formation by cultured melanoma cells after treatment with isolated melanosomes.

Melanosomes isolated from a subcutaneous Harding-Passey mouse melanoma and purified by density gradient centrifugation were labelled in vitro with 14C-tyrosine or 3H-dihydroxyphenylalanine in the melanin portion. Incubation of monolayer cultures of Harding-Passey melanoma cells during exponential growth phase (wherin cells contained relatively few melanosomes) with isolated and labelled melanosomes during a time-period of up to 3 days resulted in rapid cellular uptake of label (reaching a saturation level after about half a day). Following a lag period of several hours, the melanin content rose near-linearly in the course of 3 days. Comparison of curves of uptake of radioactivity and melanin concentration indicates that the latter rise is due primarily to newly formed melanin. Ultrastructural studies revealed a strikingly increased number of melanosomes in melanosome-treated cells. Some of these appeared to be the result of phagocytotic uptake. In fact, invagigations of the plasma membrane containing exogenous melanosomes were observed. Since most intracellular melanosomes were localized directly in the cytoplasmic matrix, dissolution of the phagosome membrane appeared to have taken place. Aggregates of melanosomes either surrounded by a membrane or free in the cytoplasm were also observed. These bodies might represent phagolysosomes or/and centres for the formation of new melanosomes. The combined biochemical and ultrastructural findings suggest stimulated melanogenesis induced by phagocytized melanosomes.

Animals↗
PubMedDate not supplied

Proteomic analysis of early melanosomes: identification of novel melanosomal proteins.

Melanin is a heterogeneous biopolymer produced only by specific cells termed melanocytes, which synthesize and deposit the pigment in specialized membrane-bound organelles known as melanosomes. Although melanosomes have been suspected of being closely related to lysosomes and platelets, the total number of melanosomal proteins is still unknown. Thus far, six melanosome-specific proteins have been identified, and the challenge is to characterize the complete proteome of the melanosome to further understand its mechanism of biogenesis. In this report, we used mass spectrometry and subcellular fractionation to identify protein components of early melanosomes. Using this approach, we have identified all 6 of the known melanosome-specific proteins, 56 proteins that are shared with other organelles, and confirmed the presence of 6 novel melanosomal proteins using western blotting and by immunohistochemistry.

Blood Platelets↗
PubMed1982

Characterization of structural properties for morphological differentiation of melanosomes: II. Electron microscopic and SDS-PAGE comparison of melanosomal matrix proteins in B16 and Harding Passey melanomas.

This study compared the qualitative and quantitative differences of the melanosomal matrix proteins between the ellipsoidal-lamellar melanosomes of B16 melanoma and the spherical-granular melanosomes of Harding Passey (HP) melanoma by electron microscopy and SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). The purity of the melanosomes at each step of the sample preparations was assayed by % distribution of cytoplasmic marker enzymes and proteins. Characterization of the melanosomal matrix was carried out in material purified by the 2 steps of sucrose density gradient ultracentrifugation and membrane dissociation with BRIJ-35. Eighty-three percent of the matrix proteins were solubilized from both B16 and HP by guanidine HCl (GH), which also caused marked ultrastructural changes in different ways. The inner matrix of HP completely disintegrated into fine grains whereas the matrix of B16 showed a great loss in electron density and was degraded, though its basic framework remained unchanged. Under SDS-PAGE, the melanosomal matrix was dissociated into 14 polypeptide bands with respect to size and charge density, 10 of which (63-85% of total proteins solubilized by GH) were common to both B16 and HP. A significant quantitative difference was noted in the relative amounts of these common proteins. The remaining 4 were unique in molecular weight to each form of melanosome though their relative amounts were low. It is suggested that these chemical differences, i.e., quantitative and qualitative, of the matrix proteins are responsible for the ultrastructural differentiation of melanosomes.

Animals↗
PubMed1984

Characterization of melanogenesis and morphogenesis of melanosomes by physicochemical properties of melanin and melanosomes in malignant melanoma.

This study elucidates the nature of melanogenesis in B16 and Harding-Passey (HP) mouse melanomas producing melanin and melanosomes of different color and fine structure, i.e., brown-black eumelanosome-like B16 granules and reddish brown pheomelanosome-like HP granules, and compares them with "typical" 3,4-dihydroxyphenylalanine (DOPA) and sepia eumelanins and sepia eumelanosomes. The melanin content of B16 melanosomes was more than three times higher than that of HP melanosomes. The content of free and protein-bound DOPA and 5-S-cysteinyldopa varied greatly in B16, HP, and sepia melanosomes and was unrelated to melanin content. Chemical analysis of the eumelanin: pheomelanin ratio in melanosomes and elemental analysis of isolated melanin showed that B16 and HP melanins are primarily eumelanic, with a higher ratio of pheomelanic component in HP melanin. The spectra of electron spin resonance and IR and X-ray small-angle scattering of B16 and HP melanins were basically similar to those of sepia and DOPA melanins. B16, HP, and DOPA melanins were dissolved in aqueous NH3, while sepia melanin was dissolved to a far lesser extent. It was concluded that both B16 and HP melanomas are primarily involved in eumelanogenesis, although the fine structure of their melanosomes is entirely different, and that the marked color difference in the two melanosomes is related to a difference in the absolute content of eumelanin, the presence of a small amount of pheomelanin, and the mode of chemical bindings of melanin to structural proteins. In contrast to normal skin and hair, melanosome morphogenesis may not directly correspond to melanogenesis type in malignant melanoma.

Animals↗
PubMed1999

The pink-eyed dilution locus controls the biogenesis of melanosomes and levels of melanosomal proteins in the eye.

The pink-eyed dilution (p) locus is known to control the quantity of melanin pigment made within melanocytes and retinal pigment epithelium (RPE) in the eye. We have examined the effects of several mutant allele combinations at the murine p locus on the number and morphology of melanosomes in choroidal melanocytes and RPE cells as well as on the levels of four proteins known to be present within melanosomes: tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) and lysosome-associated membrane protein-1 (LAMP-1). By electron microscopy, we observed a modest diminution in the size and number of choroidal melanosomes in pbs/pJ mice but a more dramatic decrease in the RPE in comparison with wild-type P/P mice. By contrast, a drastic reduction in melanosome size and number was present in the choroid and RPE of pun/pun and p6H/pcp mice, and in the RPE of p6H/pcp mice, melanosomes were essentially undetectable. In wild-type mice, levels of tyrosinase, TRP-1 and TRP-2 were high at birth and showed a second peak of expression at 10-14 days of age, declining to undetectable levels by 42 days. All three mutant allele combinations reduced the levels of these melanosomal proteins with the relative severity of effects being p6H/pcp>pun/pun>pbs/pJ. In the null p6H/pcp mice, levels of these proteins were extremely low at birth, no postnatal peak was observed, and levels declined to undetectable by 14 days. Levels of LAMP-1 in wild-type mice rose initially and then declined whereas in the mutant mice, levels decreased gradually from birth. Higher levels of LAMP-1 were observed in each of the mutants than in the wild-type mice at 21 days of age. Our results demonstrate that mutations at the p locus affect the size, number, shape and contents of melanosomes, implicating the p gene product in the normal biogenesis of this organelle.

Animals↗
PubMed2002

Rab27b association with melanosomes: dominant negative mutants disrupt melanosomal movement.

The movement of melanosomes from post-Golgi compartments to the periphery of melanocytes is known to be regulated by factors including myosin Va and at least one Rab protein, Rab27a. Mutations in the genes encoding either protein in the mouse result in a hypopigmented phenotype mimicking the human disease Griscelli syndrome. Rab27b and Rab27a share 72% identity and they belong to the same melanocyte/platelet subfamily of Rab proteins. Rab27a orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. By contrast, the function of Rab27b has remained elusive. In this study, we found that Rab27b mRNA is present in melanocytes and demonstrated the intrinsic GTPase activity of Rab27b protein. We explored the function of Rab27b by overexpression of two dominant negative mutants as well as the wild-type Rab27b in melan-a melanocytes. Green-fluorescent-protein-tagged Rab27b colocalizes with the melanosome marker tyrosinase-related protein 1 and with myosin Va at the cell periphery, whereas Rab27b mutants do not decorate melanosomes, and melanosomes in these mutant transfected cells redistribute from cell periphery to the perinuclear region. Furthermore, transient overexpression of the dominant negative forms of Rab27b caused diminution in both numbers and length of dendrites of melan-a cells. Our results suggest that Rab27b may regulate the outward movement of melanosomes and the formation or maintenance of dendritic extensions in melanocytes.

Animals↗
PubMed1997

The predominant defect in dilute melanocytes is in melanosome distribution and not cell shape, supporting a role for myosin V in melanosome transport.

Mice with mutations at the dilute locus, which encodes the heavy chain of a type V unconventional myosin, exhibit a reduction in coat colour intensity. This defect is thought to be caused by the absence in dilute melanocytes of the extensive dendritic arbor through which these cells normally deliver pigment-laden melanosomes to keratinocytes. The data on which this conclusion has been based can also be explained, however, by a defect in the outward transport of melanosomes within melanocytes of normal shape. To resolve this question, we compared the shape and pigment distribution within melanocytes present in primary cultures prepared from the epidermis of C57BL/6J pups that were either wild type (D/D) at dilute or homozygous for the dilute null allele d120J. These same comparisons were also performed on melanocytes in situ, where antibodies to the membrane tyrosine kinase receptor cKIT were used to visualize melanocyte cell shape independent of pigment distribution. Wild type melanocytes were found to be dendritic and to have melanosomes distributed throughout their dendrites both in vitro and in situ. Mutant melanocytes were also found to be dendritic in both cases, but their melanosomes were highly concentrated in the cell body and largely excluded from dendrites. We conclude, therefore, that the predominant defect in dilute melanocytes is in melanosome distribution, not cell shape. These results argue that the myosin V isoform encoded by the dilute locus functions in dendritic extensions to move melanosomes from their site of formation within the cell body to their site of intercellular transfer at dendritic tips. This conclusion is consistent with our recent demonstration by immunolocalization that the dilute myosin V isoform associates with melanosomes in mouse melanocytes.

Animals↗
PubMed1993

Characterization of melanosome-associated proteins by establishment of monoclonal antibodies and immunoscreening of a melanoma cDNA library through an anti-melanosome antibody.

Monoclonal antibodies against melanosomal components (human melanosome specific antigens; HMSAs) have been developed in our laboratory. HMSA-1-4 recognizes structural matrix proteins of melanosomes. HMSA-5 is identical to TRP-1, equivalent to the b (brown) locus of murine melanocytes and expressed in early stages of melanosomal maturation. HMSA-6 is a protein associated with melanosomes but its role is still unclarified, and HMSA-7 is identical to the lysosomal protein CD63. We have also recently identified p90 calnexin-like, Ca(2+)-binding protein p97 melanotransferrin, and p64 beta-D-galactosidase-like protein associated with melanosomes through immunological screening of our melanocytes (melanoma cells) cDNA library. Approximately 150 genes and 60 loci are known to influence eye, skin and hair colour in mammals. Tyrosinase is a rate-limiting enzyme responsible for melanin synthesis. In addition, tyrosine-related proteins (TRPs) and their genes have been identified and cloned. Tyrosinase and TRPS (e.g., TRP-1; b-locus protein identical to HMSA-5 and TRP-2; dopachrome tautomerase) are synthesized according to underlying genetic programmes, and are up- and/or down-regulated to create various forms of abnormal melanin pigmentation. We herein propose the importance of investigating the role of non-tyrosinase related proteins such as those which we have recently identified.

Animals↗
PubMed2005

MART-1 is required for the function of the melanosomal matrix protein PMEL17/GP100 and the maturation of melanosomes.

More than 125 genes that regulate pigmentation have been identified to date. Of those, MART-1 has been widely studied as a melanoma-specific antigen and as a melanosome-specific marker. Whereas the functions of other melanosomal proteins, such as tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, and Pmel17, are known, the function of MART-1 in melanogenesis, is unclear. A role for MART-1 in pigmentation is expected because its expression pattern and subcellular distribution is quite similar to the other melanosomal proteins and usually correlates with melanin content. We investigated the function of MART-1 using a multidisciplinary approach, including the use of siRNA to inhibit MART-1 function and the use of transfection to re-express MART-1 in MART-1-negative cells. We show that MART-1 forms a complex with Pmel17 and affects its expression, stability, trafficking, and the processing which is required for melanosome structure and maturation. We conclude that MART-1 is indispensable for Pmel17 function and thus plays an important role in regulating mammalian pigmentation.

Animals↗
PubMed2001

A melanosome-associated monoclonal antibody J1 recognizes luminal membrane of prelysosomes common to biogenesis of melanosomes and lysosomes.

Melanogenesis cascade may be directly or indirectly linked to the dynamics of endosome-lysosome biogenesis. This study aims to identify how and to what extent the endosome-lysosome system is involved in melanosome biogenesis, by utilizing a novel melanogenesis marker, J1, which we identified in the process of developing monoclonal antibodies (MoAbs) against human melanosomes. The antigenic epitope of MoAb J1 was expressed by all of the melanotic and nonmelanotic cells examined. It was expressed primarily by granular structures located in regions proximal to the Golgi complex. Most of MoAb J1 positive granules were co-stained with melanogenic markers, tyrosinase or tyrosinase-related protein (TRP-1). The epitope of MoAb J1 was also coexpressed by most, but not all, of LGP85 (a lysosomal marker) positive granules in both melanoma and non-melanoma cells, indicating that MoAb J1 recognizes a subset of lysosomal vesicles. MoAb J1 did not, however, react with vesicles with late/early (syntaxin 8/ EEA1) endosomal markers. Further examination using fluorophore-labeled pepstatin, a marker of lysosomal luminal content, confirmed that MoAb J1 specifically recognizes the luminal surface of lysosomes. These results indicate that MoAb J1 possesses an antigen epitope that is expressed in the luminal component of prelysosomal granules which are involved in the biogenesis cascade common to both melanosomes and lysosomes. We suggest that tyrosinase family protein, tyrosinase and TRP-1 are transported to melanosomes from TGN via these prelysosomal granules after being transiently transported to late endosomes.

Animals↗
PubMed1981

Freeze-fracture study of melanosomes in mammalian epidermis and the distribution of intramembrane particles in the melanosome membrane.

The ultrastruct of melanosomes in mammalian epidermis has been studied using the freeze-fracture technique. The internal structure of the melanosome, as seen in cross-fractured granules, consists of a very fine particulate matter. There is no evidence of an internal membranous structure in melanosomes, indicating that the internal matrix which is characteristic of ultra-thin sectioned material most likely represents cross-linked protein fibers. Fracture "en-face' of the melaosome limiting membrane reveals a random distribution of intramembrane particles on both the P- and E-faces of the membrane. There is a significantly higher density of IMPs on the P-face. The IMPs of the melanosome membrane may be involved in (a) selective passage of ions through the membrane, (b) membrane transducing events, and/or (c) anchoring sites for cytoplasmic fibrils and microfilaments.

Animals↗
PubMed1996

DOPA-negative melanocytes in the outer root sheath of human hair follicles express premelanosomal antigens but not a melanosomal antigen or the melanosome-associated glycoproteins tyrosinase, TRP-1, and TRP-2.

It is believed that DOPA-negative melanocytes in the outer root sheath of the human hair follicle are activated, become identifiable by DOPA staining, and migrate into the epidermis during the repigmenting phase of vitiligo. These cells are difficult to identify, however, and otherwise have not been characterized. These cells are readily identified by immunofluorescence, immunohistochemistry, and immunoelectronmicroscopy using the antibodies NKI/beteb and A4F11, which recognize premelanosome-related antigens. The majority of the outer root sheath melanocytes were found in the mid to the upper portion of the hair follicle. Double staining revealed that these cells were distinct from HLA-DR-bearing dendritic cells. Further immunohistochemical investigation using alpha-PEP-7, alpha-PEP-1, or TMH-1 and alpha-PEP-8 antibodies revealed that outer root sheath melanocytes cannot be identified by antibodies to tyrosinase, TRP-1, or TRP-2, respectively. These cells also did not react with HMB45 antibody, which recognizes a melanosome-associated cytoplasmic antigen. We believe that the inactive outer root sheath melanocytes contain some of the early structural proteins but not any of the enzymatic proteins necessary for melanogenesis. Therefore, activation is the process whereby outer root sheath melanocytes acquire all of the structural and enzymatic proteins necessary for melanogenesis.

Adult↗