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Melanosomes are specialized membrane-bound organelles that synthesize and organize melanin, ultimately providing color to the skin, hair, and eyes. Disorders in melanogenesis and melanosome transport are linked to pigmentary diseases, such as Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, and Griscelli syndrome. Clinical cases of these pigmentary diseases shed light on the molecular mechanisms that control melanosome-related pathways. However, only an improved understanding of melanogenesis and melanosome transport will further the development of diagnostic and therapeutic approaches. Herein, we review the current literature surrounding melanosomes with particular emphasis on melanosome membrane transport and cytoskeleton-mediated melanosome transport. We also provide perspectives on melanosome regulatory mechanisms which include hormonal action, inflammation, autophagy, and organelle interactions.

Uveal melanoma is an aggressive cancer which has a high percentage recurrence and with a worse prognosis. Identify the potential prognostic markers of uveal melanoma may provide information for early detection of recurrence and treatment. RNA sequence data of uveal melanoma and patient clinic traits were obtained from The Cancer Genome Atlas (TCGA) database. Co-expression modules were built by weighted gene co -expression network analysis (WGCNA) and applied to investigate the relationship underlying modules and clinic traits. Besides, functional enrichment analysis was performed on these co-expression genes from interested modules. First, using WGCNA, identified 21 co-expression modules were constructed by the 10975 genes from the 80 human uveal melanoma samples. The number of genes in these modules ranged from 42 to 5091. Found four co -expression modules significantly correlated with three clinic traits (status, recurrence and recurrence Time). Module red, and purple positively correlated with patient's life status and recurrence Time. Module green positively correlates with recurrence. The result of functional enrichment analysis showed that the module magenta was mainly enriched genetic material assemble processes, the purple module was mainly enriched in tissue homeostasis and melanosome membrane and the module red was mainly enriched metastasis of cell, suggesting its critical role in the recurrence and development of the disease. Additionally, identified the hug gene (top connectivity with other genes) in each module. The hub gene SLC17A7, NTRK2, ABTB1 and ADPRHL1 might play a vital role in recurrence of uveal melanoma. Our findings provided the framework of co-expression gene modules of uveal melanoma and identified some prognostic markers might be detection of recurrence and treatment for uveal melanoma.

Background Malignant melanomas are intrinsically resistant to many conventional treatments, such as radiation and chemotherapy, for reasons that are poorly understood. Here we propose and test a model that explains drug resistance or sensitivity in terms of melanosome dynamics. Methods The growth and sensitivity to cisplatin of MNT-1 cells, which are melanotic and enriched with mature stage III and IV melanosomes, and SK-MEL-28 cells, which have only immature stage I and II melanosomes, were compared using clonogenic assays. Differences in pigmentation, melanosome stages, melanosome number, and cellular structures in different cell lines in response to various treatments were examined by electron microscopy. The relative numbers of melanosomes of different stages were compared after treatment with 1-phenyl-2-thiourea. The relationship between drug transporter function and endogenous melanogenic toxicity was assessed by treating cells with the cyclosporin analog PSC-833 and by assessing vacuole formation and cell growth inhibition. All statistical tests were two-sided. Results Endogenous melanogenic cytotoxicity, produced by damaged melanosomes, resulted in pronounced cell growth inhibition in MNT-1 cells compared with amelanotic SK-MEL-28 cells. The sensitivity to CDDP of MNT-1 cells was 3.8-fold higher than that of SK-MEL-28 cells (mean IC(50) for SK-MEL-28 and MNT-1 = 2.13 microM and 0.56 microM, respectively; difference = 1.57 microM, 95% confidence interval = 1.45 to 1.69; P = .0017). After treatment with 6.7 microM CDDP for 72 hours, the number of stage II-III melanosomes in surviving MNT-1 cells was 6.8-fold that of untreated cells. Modulation of MNT-1 cells to earlier-stage (II, II-III, III) melanosomes by treatment with the tyrosinase inhibitor 1-phenyl-2-thiourea dramatically increased CDDP resistance. Furthermore, PSC-833 principally suppressed MNT-1 melanotic cell growth via an elevation of autophagosome-like vacuolar structures, possibly by inhibiting melanosome membrane transporters. Conclusions Melanosome dynamics (including their biogenesis, density, status, and structural integrity) regulate the drug resistance of melanoma cells. Manipulation of melanosome functions may be an effective way to enhance the therapeutic activity of anticancer drugs against melanoma.

Tyrosinase activity, synthesis, mRNA, and its posttranslational processing were compared in hair follicular melanocytes of the C3HHeAvy mouse during eumelanogenesis and phaeomelanogenesis. Tyrosinase activity was increased during eumelanogenesis; this increase was accompanied by an increase in tyrosinase synthesis. Tyrosinase activity was also increased during phaeomelanogenesis, but only to a peak level that was 50% of that during eumelanogenesis. However, tyrosinase synthesis and mRNA levels were the same in follicles during eumelanin and phaeomelanin synthesis. The lower level of tyrosinase activity is, therefore, presumably due to posttranslational regulation. Less tyrosinase was associated with the particulate fraction during phaeomelanogenesis than during eumelanogenesis. Glycosylation of tyrosinase during phaeomelanogenesis was also reduced and may be the mechanism of control. Bromo-adenosine 3,5-cyclic monophosphate sodium salt increased glycosylation in both eumelanin and phaeomelanin, producing follicles; but this did not result in an increased uptake of tyrosinase onto the melanosome membrane. Therefore, although cAMP increased glycosylation of tyrosinase, the uptake of tyrosinase by the melanosome membrane appeared to be regulated by other systems that are limiting during phaeomelanogenesis, resulting in a lower level of tyrosinase activity.

In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end–tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp–microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end directly by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p–Kar9p–Bim1p complex in yeast. We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

Patients with the autosomal recessive Griscelli-Pruniéras syndrome type II are immunologically impaired and have an unusual silvery-grey hypopigmented colour of scalp hair, eyelashes and eyebrows but no noteworthy pigmentary abnormalities of the skin. In most Griscelli patients, the RAB27A gene, which encodes a small GTPase that is associated with the melanosome membrane in melanocytes, is mutated. Here we discuss a genomic RAB27A deletion found in a 21-month-old Moroccan Griscelli patient. Additionally, we provide evidence that the loss of functional Rab27a in melanocytes of this Griscelli patient is partially compensated by the up-regulation of Rab27b, a homologue of Rab27a. By real-time quantitative PCR and western blot analysis, we found that Rab27b mRNA and protein, expressed at low levels in normal human melanocytes, is significantly up-regulated in melanocytes derived from this patient. Our immunofluorescence and yeast two-hybrid screening studies reveal that Rab27b can form a tripartite complex on the melanosome membrane with Melanophilin, a Rab27a effector, and protein products of Myosin Va transcripts that contain exon F. Our data suggest that up-regulated Rab27b in melanocytes of the Griscelli patient can partially take over the function of Rab27a, which could explain the fact that this patient had an evenly pigmented skin and was able to tan.

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, alpha, beta, delta, epsilon and zeta isoforms of PKC are expressed, but uniquely PKC-beta activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-beta phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-beta, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-beta, revealed abundant PKC-beta, whereas PKC-alpha was not detected when immunoblot analysis was performed using antibody against PKC-alpha. Activation of PKC in melanocytes increased the level of PKC-beta co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-beta in the presence of UV-activated 1, 1'-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-beta and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-beta on the melanosome membrane, allowing PKC-beta to phosphorylate tyrosinase.

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

Melanosomes are morphologically and functionally unique organelles within which melanin pigments are synthesized and stored. Melanosomes share some characteristics with lysosomes, but can be distinguished from them in many ways. The biogenesis and intracellular movement of melanosomes and related organelles are disrupted in several genetic disorders in mice and humans. The recent characterization of genes defective in these diseases has reinvigorated interest in the melanosome as a model system for understanding the molecular mechanisms that underlie intracellular membrane dynamics.

Several melanosome membrane proteins have been identified, forming a family of proteins known as tyrosinase related proteins. Human TRP-1/gp75 is sorted to melanosomes through the endoplasmic reticulum and Golgi complex to the endocytic pathway, directed by a sorting signal located in the cytoplasmic tail. This hexapeptide cytoplasmic sequence, which is conserved in the tyrosinase related protein family and through vertebrate evolution, was shown to act also as a sorting signal in mouse gp75, confirming that its sorting and cellular retention function is conserved between human and mouse. The cytoplasmic tail influenced the rate and efficiency of intracellular transport of gp75 from the endoplasmic reticulum to the cis-Golgi. Deletion of 33 or 27 amino acids from the carboxyl end of the 38 amino acid cytoplasmic tail of gp75 caused retention and rapid degradation of the truncated gp75 in the endoplasmic reticulum. This defective movement could be fully corrected by extending the truncated tail with the unrelated cytoplasmic tail of the low density lipoprotein receptor. Thus, the cytoplasmic tail of mouse gp75 not only determines sorting to the endocytic/melanosomal compartment, but also controls export from the endoplasmic reticulum to Golgi.

The melanosome is an organelle specialized for melanin synthesis that is derived from the endocytic pathway. Several melanosome membrane proteins have been identified, forming a family of proteins known as tyrosinase-related proteins. Two members of this family, tyrosinase and gp75, are well-characterized melanocyte differentiation antigens. Our previous studies have shown that gp75, the mouse brown locus protein, is sorted to melanosomes along the endocytic pathway, directed by a hexapeptide sorting signal located in the cytoplasmic tail. In this study, we report the unexpected finding that a portion of gp75 is secreted. Substantial levels of secretory gp75 were detected in melanocytic cells. Cell surface expression of gp75 was also detected, representing 2% of cellular gp75. Characterization of secretory gp75 cells showed that it is: (i) a truncated form that lacks the transmembrane region, the cytoplasmic tail where the endosomal sorting signal is located, and a small portion of the lumenal domain; (ii) more extensively glycosylated than endocytic/melanosomal gp75, containing trans-Golgi processed sugar residues; and (iii) generated post-translationally in an acid sensitive compartment after processing in the trans-Golgi, and secreted rapidly after generation. Thus, these endocytic/melanosomal membrane proteins can be processed to abundant secretory forms, probably in an endocytic compartment through a potentially novel secretory pathway.

In mammals, pigments are made by melanocytes within a specialized organelle, the melanosome. Mature, pigment-laden melanosomes are then transferred to keratinocytes to drive the visible pigmentation of the animal's hair and skin. The dilute suppressor (dsu) locus encodes an extragenic suppressor of the pigmentation defect exhibited by mice lacking myosin Va (i.e. dilute mice). We recently showed that melanoregulin, the product of the dsu locus, functions as a negative regulator of a shedding mechanism that drives the intercellular transfer of melanosomes from the melanocyte to the keratinocyte. Here we address melanoregulin's localization within the melanocyte, as well as the molecular basis for its localization. First, we confirm and extend recently published results using exogenous, GFP-tagged melanoregulin by showing that endogenous melanoregulin also targets extensively to melanosomes. Second, using site-directed mutagenesis, metabolic labeling with H(3)-palmitate, and an inhibitor of palmitoylation in vivo, we show that the targeting of melanoregulin to the limiting membranes of melanosomes in melanocytes and lysosomes in CV1 cells depends critically on the palmitoylation of one or more of six closely-spaced cysteine residues located near melanoregulin's N-terminus. Finally, using Fluorescence Recovery after Photobleaching (FRAP), we show that melanoregulin-GFP exhibits little if any tendency to cycle in and out of the melanosome membrane. We conclude that multiple palmitoylation serves to stably anchor melanoregulin in the melanosome membrane.

Our current understanding of the molecular events governing pigmentation has been largely guided by identifying over 120 genes that are altered in human patients and animals with abnormal pigmentation. Most of these genes encode melanogenic enzymes, transporters, receptors or transcription factors that are expressed only in pigment cells or that function in only limited additional cell types. The fact that animals survive with mutations in these known genes indicates a priori that they are not required for viability. Undoubtedly, pigmentation must also require additional genes that regulate essential functions in many cell types, and which therefore may not be represented among viable animals with altered pigmentation. In a recent issue of PLoS Genetics, Ganesan and colleagues report a large number of candidate genes in this category that surprisingly drive pigment cells towards the dark side. To identify genes involved in pigmentation in an unbiased way, the authors used a simple but novel high-throughput screen. They assayed for decreased melanin content in a human melanoma line that was transduced with a library consisting of small interfering RNA (siRNA) duplexes directed to each of over 21,000 unique human genes. The cell line, MNT-1, has been widely used as a model for eumelanin pigmentation and melanosome formation. Importantly, these cells constitutively release melanin into the medium, such that all of their melanosomes turn over in a matter of days. Thus, a block in the formation of new pigment - by interfering with either melanosome biogenesis or expression of melanogenic genes - would result in a decrease in melanin content within 4–5 days of culture, well within the time-frame of siRNA-mediated effects. By quantifying melanin content spectroscopically and controlling for loss of cell viability, Ganesan et al. identified at least 92 genes for which a siRNA-mediated reduction in expression consistently decreased melanin content by more than 2 standard deviations from the mean. A rigorous series of controls were used to ensure that the effects were reproducible, paralleled by loss of expression of the target gene, not due to off-target effects, and - for at least some of the siRNAs - reproducible in primary human melanocytes. As verification for the approach, the authors showed that pigmentation was substantially reduced by siRNAs to 13 of 68 genes previously shown to regulate melanogenesis at the cellular level, including enzymes (e.g. tyrosinase), transporters (e.g. OCA2/ pink-eyed dilute), trafficking regulators (e.g. subunits of Hermansky-Pudlak Syndrome-associated complexes BLOC-1 and BLOC-2), signaling proteins and receptors (e.g. endothelin receptor and BMP1), and transcription factors (e.g. ZIC2). While the screen successfully identified known genes, the novel hits in the screen revealed several surprising features of the cellular and molecular control of pigmentation. The first surprising result was that half of the identified genes influenced expression of tyrosinase, the limiting enzyme in melanin synthesis. The target genes in this category spanned a broad array of protein classes, including nuclear transcription factors (e.g. nucleoplasmin 3), trafficking regulators (e.g. the small GTPases Rab4a and Arl4a), metabolic enzymes (e.g. methionine sulfoxide reductase), proteinase inhibitors (serpinB1 and serpinB2), and lipid binding proteins of unknown function (e.g. PLEKHA3). Depletion of half of these targets affected the level of tyrosinase mRNA expression, and depletion of some (e.g. Rab4a) resulted in lysosomal degradation of the tyrosinase enzyme as judged by restoration of tyrosinase upon lysosome neutralization. Thus, this list provides a large group of potential regulators of tyrosinase trafficking, gene expression, and activity. Notably, most siRNAs that decreased tyrosinase protein levels also down-regulated mRNA levels for the transcription factor MITF. This suggests a previously unanticipated regulatory feedback loop in which tyrosinase activity might be required to maintain MITF-dependent melanocyte differentiation. Defining the mechanistic basis for this feedback loop might provide insights into potential regimens for treatment of pigmentary diseases. Perhaps the most intriguing hits identified in this screen were a group of genes known to encode regulators of macroautophagy. Macroautophagy, or self-eating, is an essential cellular process in which cytosolic components are selectively entrapped within a unique double membrane structure called an autophagosome. The contents of the autophagosome are then degraded upon fusion with lysosomes. All cells undergo constitutive macroautophagy at a basal level, but macroautophagy is highly induced in response to certain stimuli such as starvation and is dysregulated in numerous diseases including cancer and neurodegeneration (Mizushima et al., 2008). Autophagosome formation and fusion with lysosomes are mediated by a cascade of factors that were first discovered in the yeast, Saccharomyces cerevisiae, and that are conserved throughout metazoan evolution (Xie and Klionsky, 2007). Most of these factors are thought to be dedicated to autophagy alone, although some have additional known functions in cell signaling or trafficking. In the siRNA screen or subsequent directed siRNA analyses, Ganesan et al. identified several genes that are thought to regulate nascent autophagosome formation. Four identified genes are homologues of core machinery components for nascent autophagosome formation in yeast, and include WIPI1 (homologous to yeast Atg18), Beclin1 (homologous to Atg6), and LC3-C and LC3-A (both homologues of yeast Atg8). A fifth identified gene, GPSM1/ ASG3, is thought to stimulate autophagy in colon carcinoma cells (Pattingre et al., 2003). Ganesan et al support a role for macroautophagy regulators in animal pigmentation by showing that mice engineered to lack beclin-1 have dilute coat color. Together, the data suggest the intriguing possibility that pigment cells employ a group of proteins involved in the early stages of autophagy to effect pigmentation. Although Ganesan et al did not test for effects of their siRNA hits on melanosome morphology, maturation or content, the most straightforward hypothesis based on their results is that pigment cells divert the machinery involved in autophagosome formation to facilitate melanosome formation. Consistent with this idea, the authors provide evidence that LC-3 and Apg5, two autophagy effectors, might accumulate on the membrane of Pmel17-containing stage II melanosomes. At least two potential roles for these effectors can be envisioned. First, they might facilitate membrane deposition required to segregate stage II melanosomes from the early endosomal system. Second, they might function at the level of multivesicular endosomes, which are intermediates in both stage II melanosome maturation and biosynthetic delivery of mature melanosome membrane proteins. Components of the machinery that regulates multivesicular body formation are also required for autophagy (Rusten and Simonsen, 2008); whether the autophagy machinery inversely regulates multivesicular body formation in metazoans is not yet known, but could potentially account for its requirement in pigmentation. Another intriguing possibility is that the specific autophagy components identified in this screen also function in processes unrelated to autophagy. For example, Atg18 - the yeast orthologue of WIPI1 - is one of the few known proteins that bind to the doubly phosphorylated phospholipid, phosphatidylinositol(3,5)-bisphosphate (PtdIns(3,5)P2), and has been shown to function in membrane retrieval from the vacuole - the yeast equivalent of the lysosome (Dove et al., 2004). PtdIns(3,5)P2 metabolism is directly altered in a form of Charcot-Marie Tooth syndrome that is associated with hypopigmentation (Chow et al., 2007), implying an important role for PtdIns(3,5)P2 in membrane transport processes required for melanization (see (Marks, 2008)). If WIPI1 also binds PtdIns(3,5)P2, it might therefore regulate melanosome formation in an autophagy-independent manner. Another identified hit, GPSM1, is also indirectly linked to a previously known regulator of melanosome biogenesis, GIPC. GPSM1 stimulates autophagy by stabilizing the GDP-bound form of the heterotrimeric G protein component, Gαi3 (Pattingre et al., 2003). Levels of Gαi3-GDP are also elevated by the activity of the GTPase activating protein, GAIP. GAIP directly interacts with GIPC, a PDZ binding protein that binds to the cytoplasmic domain of the melanosomal protein, Tyrp1, and that is thought to regulate Tyrp1 trafficking (Liu et al., 2001). It is therefore possible that GPSM1, perhaps via stabilization of Gα13-GDP and subsequent sequestration of GAIP, regulates Tyrp1 trafficking. These data are consistent with the notion that GPSM1 and WIPI1 - and perhaps other autophagy components identified in the screen - influence pigmentation in a manner independent of their function in autophagy. Regardless of the mechanisms of action that are sure to be elucidated in the future, Ganesan et al have provided us with provocative leads toward new players and pathways involved in melanization. The findings are sure to stimulate thought and new insight into the signaling and membrane trafficking pathways required for pigmentation.

Recruitment of MyoVa and the proper transport of melanosomes during pigment dispersion requires the central region of melanophilin (Mlph, 90 kDa) to bind MyoVa and the N-terminal region bound to the melanosome membrane via Rab27a. The interaction among these proteins is the key to melanosome transport. Previously, we identified and mapped for the first time the interaction between PFD4 (∼14kDa) and Mlph using the yeast 2-hybrid system (in vivo) and a biochemical assay (in vitro). PFD4 is a subunit of prefoldin (PFD, ∼87kDa), a chaperone that delivers unfolded proteins to a chaperonin for correct folding. Our in-vivo results suggest that PFD4 interacts with Mlph at the same MyoVa binding site. Here we confirm that interaction using pull-down assays and fluorescence spectroscopy; PFD4 competes with MyoVa for the Mlph binding site and residues 400-590 (putative coiled coil) of Mlph are crucial for PFD4 binding. In-vitro fluorescence anisotropy reveals interaction of fluorescein-labeled full-length Mlph with MyoVa tail or PFD4, by an increase in anisotropy and polarization values. Neither mutated A453P full-length Mlph nor the 400-590 segment caused a significant change in anisotropy when incubated with MyoVa; thus these constructs do not bind Mlph. Full-length Mlph also did not bind muscle myosin II. The MyoVa binding domain for Mlph and fragments 150-400, 300-433, 400-590, and Mlph A453P seems to be intrinsically unstructured. When we pre-incubated Mlph with PFD4 or MyoVa the circular dichroism spectrum showed that binding Mlph 150-400 and 150-590 with PFD4 and MyoVa tail possibly causes an increase in α-helix content. Support: CNPq, FAPERJ, PRONEX, CAPES (Brazil); Wellcome Trust (UK)

Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and (1)H NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. We discuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.

Rab27a and melanophilin (Mlph) are required in vivo to form a melanosome receptor for myosin Va in which Rab27a anchored in the melanosome membrane recruits Mlph, which in turn recruits myosin Va. Here, we show by reconstitution using purified proteins that Rab27a and Mlph are sufficient to form a transport complex with myosin Va in vitro. These results suggest that additional proteins are not required in vivo for assembly of the myosin Va receptor, although other proteins may associate with this tripartite complex to regulate its activity and/or to assist Rab27a in anchoring the complex to the melanosome membrane.

Vitiligo is a psychologically devastating clinical conundrum which affects approximately 1% of the general population. The exact cause of the illness is an enigma, but several hypotheses about its pathogenesis are advanced.The autoimmune hypothesis proposes an autoimmune attack against melanocytes. Although anti-melanocyte autoantibodies have been demonstrated in vitiligo, recent research casts doubt on their pathogenic role and instead supports the involvement of cell-mediated autoimmune response in the pathobiology of this disorder, which is characterized by increase of suppressor T-cells and decrease of the helper/suppressor ratio in association with the presence of type-1 cytokine secreting cytotoxic T cells in the vicinity of disappearing melanocytes.The neural hypothesis proposes that increased release of norepinephrine, a melanocytotoxin, from the autonomic nerve endings in the microenvironment of melanocytes injures these cells. Moreover, norepinephrine induces the catecholamine degrading enzyme monoamine oxidase (MAO), which favors the formation of toxic levels of hydrogen peroxide in the vicinity of melanocytes.Another theory suggests that abnormal permeability of melanosome membrane, which normally prevents the diffusion of toxic melanin precursors into the cytoplasm, may cause melanocyte damage.Phenytoin, the widely-used anticonvulsant, has been employed both topically and systemically in the treatment of some dermatological disorders. The drug has been shown to significantly suppress mitogen-induced activation of lymphocytes and cytotoxic T lymphocyte activity and to polarize the immune response toward the type-2 pathway. It also significantly decreases suppressor T cells and increases the helper/suppressor ratio.At high concentrations, the drug inhibits the release of norepinephrine and the activity of MAO. Moreover, phenytoin is suggested to interact with membrane lipids, which may promote stabilization of the membranes.The hydantoin moiety of phenytoin exerts a direct stimulatory action on melanocytes; facial hyperpigmentation is a recognized side effect of orally administered phenytoin.Altogether, the above evidence suggests that phenytoin could be therapeutically effective against vitiligo. As phenytoin stimulates collagen production and inhibits its breakdown, its concomitant use with topical steroids could prevent steroid-induced skin atrophy while potentiating the anti-vitiligo effect of these agents.

Complement and Fc receptor (FcR)-positive cells mediate effector functions of antibodies. Antibody-dependent immunity against the melanosome membrane glycoprotein gp75/tyrosinase-related protein-1 (TYRP-1) of melanocytes leads to autoimmune hypopigmentation (vitiligo) in mice. Hypopigmentation occurred in mice deficient in activating FcR containing the common gamma subunit (Fc gamma R gamma(-/-)) and in mice deficient in the C3 complement component. Mice doubly deficient in both Fc gamma R gamma and C3 did not develop hypopigmentation, suggesting that complement and Fc gamma R formed redundant mechanisms. Following passive immunization with antibody, no further adaptive immune responses were required. Chimeric Fc gamma R gamma(-/-),C3(-/-) mice reconstituted with bone marrow from either Fc gamma R gamma(-/-) or C3(-/-) mice or adoptively transferred with Fc gamma R gamma(+/-) macrophages did develop antibody-mediated hypopigmentation. Thus, either complement or macrophages expressing activating Fc gamma R can independently and alternatively mediate disease in a model of autoimmune vitiligo.

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.