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

Bowman et al. show that in melanocytes, the vSNARE VAMP7 is sorted from endosomes into tubular membrane transport carriers bound for maturing melanosomes in a complex with the tSNARE syntaxin 13 via redundant recognition of each SNARE by an AP-3–BLOC-1 super-complex., Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1–dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3–BLOC-1 super-complex.

Centrosomes duplicate only once in coordination with the DNA replication cycle and have an important role in segregating genetic material. In contrast, most cancer cells have centrosome aberrations, including supernumerary centrosomes, and this correlates with aneuploidy and genetic instability. The tumor suppressors p16 (CDKN2A) and p15 (CDKN2B) (encoded by the familial melanoma CDKN2 locus) inhibit CDK4/6 activity and have important roles in cellular senescence. p16 is also associated with suppressing centrosomal aberrations in breast cancer; however, the role of p15 in centrosome amplification is unknown. Here, we investigated the relationship between p15 and p16 expression, centrosome number abnormalities, and melanoma progression in cell lines derived from various stages of melanoma progression. We found that normal human melanocyte lines did not exhibit centrosome number abnormalities, whereas those from later stages of melanoma did. Additionally, under conditions of S-phase block, p15 and p16 status determined whether centrosome overduplication would occur. Indeed, removal of p15 from p16-negative cell lines derived from various stages of melanoma progression changed cells that previously would not overduplicate their centrosomes into cells that did. Although this study used cell lines in vitro, it suggests that, during clinical melanoma progression, sequential loss of p15 and p16 provides conditions for centrosome duplication to become deregulated with consequences for genome instability.

Melanin synthesis occurs within a specialized organelle called the melanosome. Traditional methods for measuring melanin levels rely on the detection of chemical degradation products of melanin by high-performance liquid chromatography (HPLC). Although these methods are robust, they are unable to distinguish between melanin synthesis and degradation, and are best suited to measure melanin changes over long periods of time. We developed a method that actively measures both eumelanin and pheomelanin synthesis by fate tracing [U-(13)C] tyrosine using liquid chromatography-mass spectrometry (LC-MS). Using this method, we confirmed previous reports of melanin synthesis differences between melanocytes derived from individuals with different skin color or MC1R genotype, and uncovered new information regarding the differential de novo synthesis of eumelanin and pheomelanin, also called mixed melanogenesis. We also revealed that distinct mechanisms that alter melanosomal pH differentially induce new eumelanin and pheomelanin synthesis. Finally, we revealed that the synthesis of L-DOPA, an important metabolite of tyrosine, is differentially controlled by multiple factors. Because tyrosine fate tracing is compatible with untargeted LC-MS based metabolomics, this approach enables the broad measurement of cellular metabolism in combination with melanin metabolism, and we anticipate that this approach will shed new light on multiple mechanisms of melanogenesis.

© The Author(s) 2020., Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm., This work was supported by a Medical Research Council New Investigator Award to A.N.H. (grant reference G1100063), Wellcome Trust Grant Awards (grant reference 108429/Z/15/Z to E.V.S. and 204843/Z/16/Z to A.N.H.), a Biotechnology and Biological Sciences Research Council (grant reference BB/F016956/1) and University of Nottingham funded PhD studentship awarded to C.L.R. D.A.B. was funded by Biochemical Society Vacation Studentship. L.M. is funded through the Spanish Ministry of Economy. Industry and Competitiveness (MINECO) [BIO2015-70978-R]. E.K. and T.W. are funded by DFG SPP1464, KE 447/10-2” and DFG KE 447/18-1. F.S. is funded by DFG Research Training Group, GRK 2174 Award. N.A. is supported by a PhD studentship from Taibah University, Medina, Kingdom of Saudi Arabia. W.W.K. and E.W.T. are funded by The Royal Society (Newton International Fellowship grant NF161582 to W.W.K.), European Commission (Marie Sklodowska Curie Individual Fellowship grant 752165 to W.W.K.), and Cancer Research UK (C29637/A20183 to E.W.T.). A.K.M. acknowledges financial support of the John and Lucille van Geest Foundation.

Melanin synthesis occurs within a specialized organelle called the melanosome. Traditional methods for measuring melanin levels rely on the detection of chemical degradation products of melanin by high-performance liquid chromatography. Although these methods are robust, they are unable to distinguish between melanin synthesis and degradation and are best suited to measure melanin changes over long periods of time. We developed a method that actively measures both eumelanin and pheomelanin synthesis by fate tracing [U-13C] L-tyrosine using liquid chromatography-mass spectrometry. Using this method, we confirmed the previous reports of the differences in melanin synthesis between melanocytes derived from individuals with different skin colors and MC1R genotype and uncovered new information regarding the differential de novo synthesis of eumelanin and pheomelanin, also called mixed melanogenesis. We also revealed that distinct mechanisms that alter melanosomal pH differentially induce new eumelanin and pheomelanin synthesis. Finally, we revealed that the synthesis of L-3,4-dihydroxyphenylalanine, an important metabolite of L-tyrosine, is differentially controlled by multiple factors. Because L-tyrosine fate tracing is compatible with untargeted liquid chromatography-mass spectrometry‒based metabolomics, this approach enables the broad measurement of cellular metabolism in combination with melanin metabolism, and we anticipate that this approach will shed new light on multiple mechanisms of melanogenesis.

Oculocutaneous albinism (OCA) is a heterogeneous and autosomal recessive hypopigmentation disorder, which is caused by mutations of genes involved in pigment biosynthesis or melanosome biogenesis. We have previously identified NCKX5 (also known as SLC24A5) as a causative gene for OCA type 6 (OCA6). However, the pathogenesis of OCA6 is unknown. We found that NCKX5 is localized to mitochondria, not to melanosomes. Pharmacological inhibition of mitochondrial function or NCKX exchanger activity reduced pigment production. Loss of NCKX5 attenuated Ca(2+) enrichment in melanosomes, which compromised PMEL fibril formation, melanosome maturation and pigment production. Thus, we have defined a new class of hypopigmentation attributable to dysfunctional mitochondria and an impairment of mitochondrial Ca(2+) transfer into melanosomes. Thus, it is possible that mitochondrial function could have a role in the graying of hair in older people and formation of hypopigmented lesions in vitiligo patients.

Dennis et al. analyze cycling of the v-SNARE VAMP7 during melanosome biogenesis in melanocytes. VAMP7 is targeted to and retrieved from maturing melanosomes in separate tubular carriers whose formation requires distinct BLOCs, each defective in variants of Hermansky–Pudlak syndrome., Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1–dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3–dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky–Pudlak syndrome variants.

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that motor myosin-Va, works with dynamic actin tracks, to drive long-range organelle dispersion in microtubule depleted cells. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here we show that SPIRE1/2 and formin-1 (FMN1) proteins generate actin tracks required for myosin-Va-dependent transport in melanocytes. Moreover we show that, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIRE1/2 to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this we suggest a model in which organelles and force generators (motors and track assemblers) are linked forming a cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.

Quantitative analyses of melanosome cargo localization and trafficking and of endosomal membrane dynamics in immortalized melanocytes from mouse Hermansky–Pudlak syndrome models show that BLOC-2 functions to specify the delivery of recycling endosomal cargo transport intermediates to maturing melanosomes., Hermansky–Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2–deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2–deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.

Summary In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1–8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks [2]. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering [5, 9]. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning [10, 11]., Graphical Abstract, Highlights • Microtubules are essential for centripetal, but not centrifugal, melanosome transport • Myosin-Va and a dynamic actin pool drive long-range centrifugal melanosome transport • Myosin-Va is a processive plus-end-directed motor, and not a tether, in melanocytes • Opposing myosin-Va/actin and microtubule forces regulate melanosome distribution, In animal cells, microtubules and actin are thought to regulate long- and short-range transport, respectively. Here, Evans et al. test the contribution of these systems to organelle transport using melanocyte pigment granules as a model. Surprisingly, they find that myosin-Va and dynamic actin drive long-range transport to the membrane.

SummaryNearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.

OCA2 is used as a model melanosome cargo protein to define primary sequence elements required for acidic dileucine–motif binding to adaptors AP-1 and AP-3. OCA2 must bind to AP-3 for melanosome localization. BLOC-1 is also required and thus can cooperate with either adaptor for cargo delivery to lysosome-related organelles., Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.

Cutaneous malignant melanoma is considered one of the most deadly human cancers, based on both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wild-type NRAS. NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here, we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine exchange factor (RalGEF), using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we discovered that constitutive RalGEF activation had a major impact on several malignant phenotypes, particularly anchorage-independent growth, indicating that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target.

The most valuable property of stem cells (SCs) is their potential to differentiate into many or all cell types of the body. So far, monitoring SC differentiation has only been possible after cells were fixed or destroyed during sample preparation. It is, however, important to develop nondestructive methods of monitoring SCs. Scanning ion conductance microscopy (SICM) is a unique imaging technique that uses similar principles to the atomic force microscope, but with a pipette for the probe. This allows scanning of the surface of living cells noninvasively and enables measurement of cellular activities under more physiological conditions than is possible with other high-resolution microscopy techniques. We report here the novel use of the SICM for studying SCs to assess and monitor the status of SCs and various cell types differentiated from SCs.

Although melanoma ultimately progresses to a highly aggressive and metastatic disease that is typically resistant to currently available therapy, it often begins as a benign nevus consisting of a clonal population of hyperplastic melanocytes that cannot progress because they are locked in a state of cellular senescence. Once senescence is overcome, the nevus can exhibit dysplastic features and readily progress to more lethal stages. Recent advances have convincingly demonstrated that senescence represents a true barrier to the progression of many types of cancer, including melanoma. Thus, understanding the mechanism(s) by which melanoma evades senescence has become a priority in the melanoma research community. Senescence in most cells is regulated through some combination of activities within the RB and p53 pathways. However, differences discovered among various tumor types, some subtle and others quite profound, have revealed that senescence frequently operates in a context-dependent manner. Here we review what is known about melanocyte senescence, and how such knowledge may provide a much-needed edge in our struggles to contain or perhaps vanquish this often-fatal malignancy.

Inactivation of the p53 pathway represents the most common molecular defect of human cancer. But in the setting of melanoma, a highly aggressive and invariably fatal malignancy in its advanced disseminated form, mutation/deletion of p53 is relatively rare, whereas its positive regulator ARF is often lost. Here, we show that genetic deficiency in Arf but not p53 facilitates rapid development of melanoma in a genetically engineered mouse model. This difference is accounted for, at least in part, by the unanticipated observation that, unlike fibroblasts, senescence control in melanocytes is strongly regulated by Arf and not p53. Moreover, oncogenic NRAS collaborates with deficiency in Arf, but not p53, to fully transform melanocytes. Our data demonstrate that ARF and p53, although linked in a common pathway, suppress tumorigenesis through distinct, lineage-dependent mechanisms and suggest that ARF helps restrict melanoma progression by executing the oncogene-induced senescence program in benign nevi. Thus, therapeutics designed to restore wild-type p53 function may be insufficient to counter melanoma and other malignancies in which ARF holds p53-independent tumor suppressor activity.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1–deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.

The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma.

SOX10 is required for melanocyte development and maintenance, and has been linked to melanoma initiation and progression. However, the molecular mechanisms by which SOX10 guides the appropriate gene expression programs necessary to promote the melanocyte lineage are not fully understood. Here we employ genetic and epigenomic analysis approaches to uncover novel genomic targets and previously unappreciated molecular roles of SOX10 in melanocytes. Through global analysis of SOX10-binding sites and epigenetic characteristics of chromatin states, we uncover an extensive catalog of SOX10 targets genome-wide. Our findings reveal that SOX10 predominantly engages ‘open’ chromatin regions and binds to distal regulatory elements, including novel and previously known melanocyte enhancers. Integrated chromatin occupancy and transcriptome analysis suggest a role for SOX10 in both transcriptional activation and repression to regulate functionally distinct classes of genes. We demonstrate that distinct epigenetic signatures and cis-regulatory sequence motifs predicted to bind putative co-regulatory transcription factors define SOX10-activated and SOX10-repressed target genes. Collectively, these findings uncover a central role of SOX10 as a global regulator of gene expression in the melanocyte lineage by targeting diverse regulatory pathways.

In mammals, >100 genes regulate pigmentation by means of a wide variety of developmental, cellular, and enzymatic mechanisms. Nevertheless, genes that directly regulate pheomelanin production have not been described. Here, we demonstrate that the subtle gray ( sut ) mouse pigmentation mutant arose by means of a mutation in the Slc7a11 gene, encoding the plasma membrane cystine/glutamate exchanger xCT [Kanai, Y. & Endou, H. (2001) Curr. Drug Metab. 2, 339-354]. A resulting low rate of extracellular cystine transport into sut melanocytes reduces pheomelanin production. We show that Slc7a11 is a major genetic regulator of pheomelanin pigment in hair and melanocytes, with minimal or no effects on eumelanin. Furthermore, transport of cystine by xCT is critical for normal proliferation, glutathione production, and protection from oxidative stress in cultured cells. Thus, we have found that the Slc7a11 gene controls the production of pheomelanin pigment directly. Cells from sut mice provide a model for oxidative stress-related diseases and their therapies.

Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol.

Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal-like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells.

International audience; The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells.

Hermansky-Pudlak syndrome (HPS) is a group of human disorders of organelle biogenesis characterized by defective synthesis of melanosomes, lysosomes, and platelet dense granules. In the mouse, at least 15 loci are associated with mutant phenotypes similar to human HPS. We have identified the gene mutated in cocoa (coa) mice, which is associated with an HPS-like mutant phenotype and thus represents a strong candidate for human HPS. Analysis of coa-mutant mice and cultured coa-mutant mouse melanocytes indicates that the normal coa gene product is involved in early stages of melanosome biogenesis and maturation.

The myelin protein P(0) has a major structural role in Schwann cell myelin, and the expression of P(0) protein and mRNA in the Schwann cell lineage has been extensively documented. We show here, using in situ hybridization, that the P(0) gene is also activated in a number of other tissues during embryonic development. P(0) mRNA is first detectable in 10-day-old embryos (E10) and is at this time seen only in cells in the cephalic neural crest and in the otic placode/pit. P(0) expression continues in the otic vesicle and at E12 P(0) expression in this structure largely overlaps with expression of another myelin gene, proteolipid protein. In the developing ear at E14, P(0) expression is complementary to expression of serrate and c-ret mRNAs, which later are expressed in sensory areas of the inner ear, while expression of bone morphogenetic protein (BMP)-4 and P(0), though largely complementary, shows small areas of overlap. P(0) mRNA and protein are detectable in the notochord from E10 to at least E13. In addition to P(0) expression in a subpopulation of trunk crest cells at E11/E12 and in Schwann cell precursors thereafter, P(0) mRNA is also present transiently in a subpopulation of cells migrating in the enteric neural crest pathway, but is down-regulated in these cells at E14 and thereafter. P(0) is also detected in the placode-derived olfactory ensheathing cells from E13 and is maintained in the adult. No signal is seen in cells in the melanocyte migration pathway or in TUJ1 positive neuronal cells in tissue sections. The activation of the P(0) gene in specific tissues outside the nervous system was unexpected. It remains to be determined whether this is functionally significant, or whether it is an evolutionary relic, perhaps reflecting ancestral use of P(0) as an adhesion molecule.

Endothelin 3 (Edn3) and its pre- ferred receptor, endothelin receptor B (Ednrb), are implicated in development, especially that of two neural-crest-derived cell lineages: melano- cytes and enteric ganglion cells. Mice and hu- mans with a null mutation at either locus can show major deficiencies in both cell types: con- genital white spotting and aganglionic megaco- lon (Hirschsprung disease in human). Numbers of early (migrating) embryonic melanoblasts are low in Ednrb ls mutant mice, while added Edn3 appears to promote the growth of melanocyte precursors in neural crest cultures. However, it is hard to assess cell differentiation in these mixed cultures, and it is not known whether Ednrb has any role in the postnatal melanocytic lineage. We have therefore studied primary cultures of neona- tal melanoblasts homozygous for the piebald (Ednrb s ) mutation. These mutant melanoblasts showed severe impairment of both net cell growth and differentiation compared to wild-type mela- noblasts. They were also unresponsive to stimula- tion of growth by cholera toxin. We have estab- lished three immortal lines of melanoblasts and one of melanocytes homozygous for Ednrb s . These immortal lines, however, had no detectable defi- ciency of growth or differentiation as judged by cell counts, induced pigmentation and immunocy- tochemistry for melanocytic markers. Consistent with this, neither Ednrb nor Edn3 mRNA was detected in 3/3 tested immortal lines of mouse melanoblasts and 5/5 lines of melanocytes, of various genotypes. We also report for the first time a method to grow immortal melanoblasts in pure culture, without feeder cells. Dev. Dyn. 1998;213:452-463. r 1998 Wiley-Liss, Inc.

Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, suggesting a developmental mutation. A report in abstract has also suggested effects on white fat and body weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet serotonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and immortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced numbers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, overproduced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimulating hormone, which normally promote melanin production. The misty gene product may be involved in adenine nucleotide metabolism or signaling.

A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.

Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis -diaminedichloroplatinum II (cDDP, cis -platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the protein-trafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocyte-stimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance ) to cDDP. Mutation of the protein-trafficking gene Hps6 , known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents.

Molecular Biology has advanced our knowledge of the individual molecular components that make up living cells. The challenge, however, is to fully understand the functional integration of these components. Our group have pioneered the development of an array of new and powerful biophysical tools based on Scanning Ion Conductance Microscopy (SICM) that allow quantitative measurements and non-invasive functional imaging of the surface of living cells down to the level of single protein molecules. Scanning ion conductance microscopy and a battery of associated innovative methods are unique among current imaging techniques, not only in spatial and temporal resolution of living cells, but also in the rich combination of imaging with other functional interrogation methods [1-3]. Recently we develop a novel class of nanoprobes based on double-barrel nanopipettes. One barrel is selectively functionalized with carbon and act as a nanoelectrode while the other one is used for ion conductance microscopy. These nanoprobes allowed simultaneous electrochemical and ion conductance imaging [4] and functional mapping of the electrochemical activity of the surface of living cells [5]. Current work in our group is directed to the use of these nanoprobes for intracellular sensing. We will present recent data demonstrating their ability to measure intracellular molecules in real time both in adherent cells and in tissue.References[1] Novak et al, Neuron, 2013, 79(6), 1067-1077.[2] Novak et al, Nat Methods, 2009, 6, 279 - 281.[3] Shevchuck et al, J Cell Biol., 2012, 197(4), 499-508.[4] Takahashi et al, Angew. Chem. Int. Ed., 2011, 50(41), 9638-9642.[5] Takahashi et al, PNAS, 2012, 109 (29), 11540-11545.

Aim of the study To investigate the efficacy of traditional Chinese medicine (TCM) in the treatment of hyperpigmentation problems, extracts of herbs selected based on traditional Chinese medical literature were screened. Materials and methods Forty extracts were extracted from 10 selected herbs using hexane, dichloromethane, methanol and water. They were then screened using melan-a cells, an immortalized non-tumorigenic mouse melanocyte cell line. Sulphorhodamine B (SRB) assay and measurement of melanin production were performed to examine the effects of the extracts as well as some natural compounds from these herbs on melanogenesis in the melan-a cells. Results The hexane and dichloromethane extracts of Angelica sinensis exhibited strong hypopigmentary effects. Conclusions Natural compounds occurring in this herb were also investigated. Among them 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol demonstrated positive effects in attenuating melanin synthesis in the cultured cells.

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.—Sviderskaya, E. V., Easty, D. J., Lawrence, M. A., Sanchez, D. P., Negulyaev, Y. A., Patel, R. H., Anand, P., Korchev, Y. E., Bennett, D. C. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.

Melanocortin-1 receptor (MC1R) and its ligands, a-melanocyte stimulating hormone (aMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan-a non agouti (a ⁄ a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200-fold increases in the pheomelanin to eumelanin ratio, and a tan-yellow color in pelletted cells., Moreover, ASIP-treated cells showed reduced proliferation and a melanoblast-like appearance, seen also in melanocyte lines from yellow (Ay ⁄ a and Mc1re ⁄ Mc1re) mice. However ASIP-YY, a C-terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP-YY inhibited the cAMP rise induced by aMSH analog NDP-MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrnmg-3J ⁄ mg-3J or Mgrn1md-nc ⁄ md-nc) also responded to both ASIP and ASIP-YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP–MC1R signaling includes a cAMP-independent pathway through attractin and mahogunin, while the known cAMP-dependent component requires neither attractin nor mahogunin., Parts of the research were supported by Wellcome Trust program grants 064583 and 078327 to D.C.B. and E.V.S.; the Japan Society for the Promotion of Science KAKENHI (grants 20790808 to T.H. and 18591262 and 20591357 to K.W. and S.I.); a Grant-in-Aid from the Japanese Ministry of Health, Labour and Welfare (K.J.), the Spanish Ministry of Education and Science BFU2006-12185 (L.M.), the South West Academic Network (A.J.D. and E.V.S.), and NIH grant DK064265 (B.Y. and G.L.M.).

Copper is a cofactor for many cellular enzymes and transporters. It can be loaded onto secreted and endomembrane cuproproteins by translocation from the cytosol into membrane-bound organelles by ATP7A or ATP7B transporters, the genes for which are mutated in the copper imbalance syndromes Menkes disease and Wilson disease, respectively. Endomembrane cuproproteins are thought to incorporate copper stably on transit through the trans-Golgi network, in which ATP7A accumulates by dynamic cycling through early endocytic compartments. Here we show that the pigment-cell-specific cuproenzyme tyrosinase acquires copper only transiently and inefficiently within the trans-Golgi network of mouse melanocytes. To catalyse melanin synthesis, tyrosinase is subsequently reloaded with copper within specialized organelles called melanosomes. Copper is supplied to melanosomes by ATP7A, a cohort of which localizes to melanosomes in a biogenesis of lysosome-related organelles complex-1 (BLOC-1)-dependent manner. These results indicate that cell-type-specific localization of a metal transporter is required to sustain metallation of an endomembrane cuproenzyme, providing a mechanism for exquisite spatial control of metalloenzyme activity. Moreover, because BLOC-1 subunits are mutated in subtypes of the genetic disease Hermansky-Pudlak syndrome, these results also show that defects in copper transporter localization contribute to hypopigmentation, and hence perhaps other systemic defects, in Hermansky-Pudlak syndrome.

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific G protein-coupled receptor exclusively localized to intracellular organelles, namely lysosomes and melanosomes. Loss of OA1 function leads to the formation of macromelanosomes, suggesting that this receptor is implicated in organelle biogenesis, however the mechanism involved in the pathogenesis of the disease remains obscure. We report here the identification of an unexpected abnormality in melanosome distribution both in retinal pigment epithelium (RPE) and skin melanocytes of Oa1-knock-out (KO) mice, consisting in a displacement of the organelles from the central cytoplasm towards the cell periphery. Despite their depletion from the microtubule (MT)-enriched perinuclear region, Oa1-KO melanosomes were able to aggregate at the centrosome upon disruption of the actin cytoskeleton or expression of a dominant-negative construct of myosin Va. Consistently, quantification of organelle transport in living cells revealed that Oa1-KO melanosomes displayed a severe reduction in MT-based motility; however, this defect was rescued to normal following inhibition of actin-dependent capture at the cell periphery. Together, these data point to a defective regulation of organelle transport in the absence of OA1 and imply that the cytoskeleton might represent a downstream effector of this receptor. Furthermore, our results enlighten a novel function for OA1 in pigment cells and suggest that ocular albinism type 1 might result from a different pathogenetic mechanism than previously thought, based on an organelle-autonomous signalling pathway implicated in the regulation of both membrane traffic and transport.

Copper is a cofactor for many cellular enzymes and transporters. It can be loaded onto secreted and endomembrane cuproproteins by translocation from the cytosol into membrane-bound organelles by ATP7A or ATP7B transporters, the genes for which are mutated in the copper imbalance syndromes Menkes disease and Wilson disease, respectively. Endomembrane cuproproteins are thought to incorporate copper stably on transit through the trans-Golgi network, in which ATP7A accumulates by dynamic cycling through early endocytic compartments. Here we show that the pigment-cell-specific cuproenzyme tyrosinase acquires copper only transiently and inefficiently within the trans-Golgi network of mouse melanocytes. To catalyse melanin synthesis, tyrosinase is subsequently reloaded with copper within specialized organelles called melanosomes. Copper is supplied to melanosomes by ATP7A, a cohort of which localizes to melanosomes in a biogenesis of lysosome-related organelles complex-1 (BLOC-1)-dependent manner. These results indicate that cell-type-specific localization of a metal transporter is required to sustain metallation of an endomembrane cuproenzyme, providing a mechanism for exquisite spatial control of metalloenzyme activity. Moreover, because BLOC-1 subunits are mutated in subtypes of the genetic disease Hermansky-Pudlak syndrome, these results also show that defects in copper transporter localization contribute to hypopigmentation, and hence perhaps other systemic defects, in Hermansky-Pudlak syndrome.

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine–based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes.

Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

Fibroblast activation protein-alpha (FAP) is a cell surface serine protease expressed at sites of tissue remodeling in embryonic development. FAP is not expressed by mature somatic tissues except activated melanocytes and fibroblasts in wound healing or tumor stroma. FAP expression is specifically silenced in proliferating melanocytic cells during malignant transformation. To study the role of FAP as a tumor suppressor, the gene for mouse fap was cloned and mutated at the catalytic domain (FAP serine mutant, FSM). We found that expression of FAP or FSM at physiologic levels in mouse melanoma cells abrogated tumorigenicity. Remarkably, the mutant form FSM lacking specific serine protease activity was a more potent tumor suppressor. Tumor rejection was not due to adaptive immune responses because RAG1-/- mice challenged with melanoma cells expressing either FAP or FSM were not tumorigenic. In in vitro assays, FAP or FSM expression restored contact inhibition, led to cell cycle arrest at G0/G1 phase, and increased susceptibility to stress-induced apoptosis. Cell death in FAP+ or FSM+ melanoma cells was readily triggered by depletion of survival factors from the media, leading to subsequent activation of caspases via the intrinsic pathway. These results show that expression of FAP is a tumor suppressor that abrogates tumorigenicity through regulation of cell growth and survival.

International audience; Protection against UV-mediated DNA damage and the onset of oncogenesis is afforded by the tanning response in which UV irradiation triggers melanocytes to increase production of melanin that is then transferred to keratinocytes. A key component of the tanning process is the UV-mediated induction of the pro-opiomelanocortin (POMC) and MC1R genes encoding the alpha-melanocyte-stimulating hormone and its receptor, respectively, which play a crucial role in pigmentation by regulating the intracellular levels of cAMP. How these genes are regulated in response to UV irradiation is not known. Here we have shown that UV-induced activation of the POMC and MC1R promoters is mediated by p38 stress-activated kinase signaling to the transcription factor, upstream stimulating factor-1 (USF-1). Importantly, melanocytes derived from USF-1 -/- mice exhibit a defective UV response and fail to activate POMC and MC1R expression in response to UV irradiation. The results define USF-1 as a critical UV-responsive activator of genes implicated in protection from solar radiation.