Melanosomes In Melanocytes Research Articles

SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models

AbstractPlatelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.

Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes

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.

Functional involvement of Rab1A in microtubule-dependent anterograde melanosome transport in melanocytes

Melanosomes are transported to the cell periphery of melanocytes by coordination between bidirectional microtubule-dependent movements and unidirectional actin-dependent movement. Although both the mechanism of the actin-dependent melanosome transport and the mechanism of the microtubule-dependent retrograde melanosome transport in mammalian skin melanocytes have already been determined, almost nothing is known about the mechanism of the microtubule-dependent anterograde melanosome transport. Small GTPase Rab proteins are common regulators of membrane traffic in all eukaryotes, and in this study we performed genome-wide screening for Rab proteins that are involved in anterograde melanosome transport by expressing 60 different constitutive active (and negative) mutants, and succeeded in identifying Rab1A, originally described as a Golgi-resident Rab, as a prime candidate. Endogenous Rab1A protein was found to be localized to mature melanosomes in melanocytes, and its functional ablation either by siRNA-mediated knockdown or by overexpression of a cytosolic form of Rab1A-GTPase-activating protein/TBC1D20 induced perinuclear melanosome aggregation. The results of time-lapse imaging further revealed that long-range anterograde melanosome movements were specifically suppressed in Rab1A-deficient melanocytes, whereas retrograde melanosome transport occurred normally. Taken together, these findings indicate that Rab1A is the first crucial component of the anterograde melanosome transport machinery to be identified in mammalian skin melanocytes.

The Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for dendrite formation in melanocytes

Vacuolar protein sorting 9 (VPS9)-ankyrin-repeat protein (Varp) has recently been identified as an effector molecule for two small GTPases-Rab32 and Rab38-in the transport of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes in melanocytes. Although Varp contains a Rab21-guanine nucleotide exchange factor (GEF) domain (i.e., VPS9 domain), since Rab21-GEF activity is not required for Tyrp1 transport, nothing is known about the physiological significance of the Rab21-GEF activity in melanocytes. Here we show by knockdown-rescue experiments that the Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for forskolin-induced dendrite formation of cultured melanocytes. We found that Varp-deficient cells are unable to extend dendrites in response to forskolin stimulation and that reexpression of wild-type Varp or a Rab32/38-binding-deficient mutant Varp(Q509A/Y550A) in Varp-deficient cells completely restores their ability to form dendrites. By contrast, VPS9 mutants (D310A and Y350A) and a vesicle-associated membrane protein 7 (VAMP7)-binding-deficient mutant were unable to support forskolin-induced dendrite formation in Varp-deficient cells. These findings indicate that the Rab21-GEF activity and Rab32/38 binding activity of Varp are required for different melanocyte functions, that is, Rab21 activation by the VPS9 domain is required for dendrite formation, and the Rab32/38 effector function of the ankyrin repeat 1 domain is required for Tyrp1 transport to melanosomes, although VAMP7-binding ability is required for both functions.

Erythema Dyschromicum Perstans in a Japanese Child

Erythema dyschromicum perstans (EDP) is asymptomatic slate-gray to blue-brown macules. Idiopathic eruptive macular pigmentation is asymptomatic brown nonconfluent macules. We describe electron microscopic studies of a 9-year-old Japanese girl with EDP. The ultrastructural figures indicated that the production of immature, small, irregular-shaped melanosomes in melanocytes and peripheral localization of melanosomes in keratinocytes caused the clinical appearance of EDP. The ultrastructural evidence distinguishes EDP from idiopathic eruptive macular pigmentation and suggests a distinct pathogenesis of the disease.

Visualization of the melanosome transfer-inhibition in a mouse epidermal cell co-culture model

Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with alphaMSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer.

Pigment Dilution Mutants from Fish Models with Connection to Lysosome-Related Organelles and Vesicular Traffic Genes

An interesting question in developmental biology is why mutations in genes with functions essential for the majority of cells produce diseases affecting only specific tissues. For example, pigment dilution disorders are often the consequence of mutations in conserved vesicular traffic genes. In Hermansky-Pudlak, Griscelli, and Chediak-Higashi pigment dilution syndromes, vesicular traffic mutations affect several organs with one characteristic in common: to carry out their functions they depend to a great extent on lysosome-related organelles (LROs), such as the melanosomes in melanocytes. Conserved multimeric complexes, present in most cell types, target proteins to lysosomes or selected LROs using transport vesicles. By studying these diseases or the model organisms that are defective in these processes, we have learned that every cell type possesses a unique way to regulate its vesicular traffic machinery and to assemble its multimeric complexes. This is accomplished by subunits from these multimeric complexes acting in a cell-specific manner. Here, we review several fish pigment dilution mutants that represent models for human vesicular traffic diseases.

Spotted hyperpigmentation: disfigured melanosomes in melanocytes and keratinocytes

Spotted hyperpigmentation: disfigured melanosomes in melanocytes and keratinocytes

What do redox metabolism, printers/lithographers, and hip replacements have to do with the prevention of melanoma?

8583 Our studies of the properties of reactive oxygen species(ROS) in melanomagenesis led us to explore the role of heavy metals in this process. A systematic experimental exploration of the effect of ROS on melanin and melanosomes in melanocytes and melanoma cells using biophysical techniques including electroparamagnetic resonance to measure free radicals and electron microscopy suggests that the conversion of melanin from an anti-oxidant and reducing agent to a pro-oxidant and photoreactive compound is an early pathogenic event that is accompanied by melanosomal disruption that serves as an ongoing organelle source of free radical generation. The involvement of selected heavy metals enhances this process and in conjunction with ultraviolet light can lead to phenotypic changes in normal human melanocytes that resemble dysplatic nevi melanocytes in culture. A re-examination of the non-genetic risk factors for melanoma has identified a large(and neglected) epidemiology that implicates heavy metals and includes markedly increased risks for melanoma in printers/lithographers(4 large studies), electrical industry workers (3 large studies), and hip replacements patients(a large meta-analyses and 2 cohort studies). High levels of cobalt are present in this latter group post-replacement that lasts for years (?indefinitely) Clinical observations (imaging studies) and studies that document the importance of metallothionein expression in primary melanoma and prognosis also offers ancillary support for the involvement of metals in the pathogenesis of melanoma Based on these considerations and the role of copper in the melanin synthesis pathway and the binding characteristics of metals to melanin we propose that the most likely culprits that accounts for a significant contribution to the etiology and pathogenesis of melanoma are Cu+2 and Co+2 and suggests that chelation may be a reasonable strategy for the prevention of melanoma. No significant financial relationships to disclose.

BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles.

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.

Rab27 and its effectors in secretory granule exocytosis: a novel docking machinery composed of a Rab27·effector complex

A small GTPase Rab27 is present on secretory granules in a wide variety of secretory cells and on melanosomes in melanocytes, and it is involved in controlling the trafficking of these organelles through interaction with a cell-type- or tissue-specific Rab27 effector(s). Slps (synaptotagmin-like proteins) and rabphilin contain an N-terminal Rab27-binding domain and C-terminal tandem C2 domains, and some of the Rab27-binding proteins have recently been shown to promote docking of Rab27-bound organelles to the plasma membrane. This mini-review presents a model for how the Rab27.effector complex controls the docking step in the trafficking of Rab27-bound organelles. Our results indicate that Slp2-a, Slp4-a/granuphilin-a and rabphilin are capable of interacting with the plasma membrane directly or indirectly, and thus that these Rab27 effectors form a bridge between Rab27-bound organelles and the plasma membrane.

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

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

Dsu functions in a MYO5A-independent pathway to suppress the coat color of dilute mice.

MYO5A is a major actin-based vesicle transport motor that binds to one of its cargos, the melanosome, by means of a RAB27A/MLPH receptor. When one of the members of this receptor-motor complex is mutated, the melanosomes clump in the perinuclear region of the melanocyte and are transferred unevenly to the developing hair, leading to a dilution of coat color. Mutation of a fourth gene, dilute suppressor (dsu), suppresses this coat color dilution. MYO5A is required for the peripheral accumulation of melanosomes in melanocytes, but its role in melanosome transfer to neighboring keratinocytes and the hair is unknown. Here, we show that MYO5A is nonessential for melanosome transfer, although pigment incorporation into the hair in MYO5A-deficient mice is uneven, probably due to the clumping of melanosomes that occurs in the perinuclear region of mutant melanocytes. We also show that dsu is caused by a loss-of-function mutation in a unique vertebrate-specific protein that appears to function in an MYO5A-independent pathway to alter pigment incorporation into the hair. Therefore, dsu identifies a unique protein involved in pigmentation of the mammalian hair.

Molecular Defects that Affect Platelet Dense Granules

Platelet dense granules form using mechanisms shared by melanosomes in melanocytes and by subsets of lysosomes in more generalized cells. Consequently, disorders of platelet dense granules can reveal how organelles form and move within cells. Models for the study of new vesicle formation include isolated delta-storage pool deficiency, combined alphadelta-storage pool deficiency, Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, thrombocytopenia absent radii syndrome, and Wiskott-Aldrich syndrome. The molecular bases of dense granule deficiency are known for the seven subtypes of HPS, as well as for Chediak-Higashi syndrome, Griscelli syndrome, and Wiskott-Aldrich syndrome. The gene products involved in these disorders help elucidate the generalized process of the formation of vesicles from extant membranes such as the Golgi.

Characterization of melanosomes in murine Hermansky-Pudlak syndrome: mechanisms of hypopigmentation.

The Hermansky-Pudlak syndrome is a genetically heterogeneous autosomal recessive disorder affecting mice and humans, which causes oculocutaneous albinism, prolonged bleeding, and in some cases, pulmonary fibrosis or granulomatous colitis. We previously demonstrated that the gene defects causing murine Hermansky-Pudlak syndrome cause blocks in melanosome biogenesis and/or trafficking in 10 Hermansky-Pudlak syndrome strains. Here, we report an in vivo quantitative analysis on five additional murine models of the Hermansky-Pudlak syndrome. We demonstrate that all strains examined here except for ashen have defects in morphogenesis, the most severely affected is sandy, muted, and buff followed by subtle gray. The ashen strain only has a defect in secretion, as indicated by retention of melanosomes in melanocytes. We document three cellular mechanisms contributing to the hypopigmentation seen in the Hermansky-Pudlak syndrome: (1) exocytosis of immature hypopigmented melanosomes from melanocytes with subsequent keratinocyte uptake; (2) decreased intramelanocyte steady-state numbers of melanosomes available for transfer to keratinocytes; and (3) accumulation of melanosomes within melanocytes due to defective exocytosis, as seen in ashen. We also report that melanosomes in the DBA/2J strain, the parental strain of the Hermansky-Pudlak syndrome strain sandy, are abnormal, indicating that aberrant biogenesis of melanosomes may play a part in the pathogenesis of pigmentary glaucoma observed in these mice.

The actin-binding domain of Slac2-a/melanophilin is required for melanosome distribution in melanocytes.

Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (DeltaABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

The roles of Rab27 and its effectors in the regulated secretory pathways.

Regulated secretory pathways are highly developed in multicellular organisms as a means of intercellular communication. Each of these pathways harbors unique store organelles, such as granules in endocrine and exocrine tissues and melanosomes in melanocytes. It has recently been shown that the monomeric GTPase Rab27 subfamily regulates the exocytosis of these cell-specific store organelles. Furthermore, genetic alterations of Rab27a cause Griscelli syndrome in humans that manifests as pigmentary dilution of the skin and the hair and variable immunodeficiency due to defects in the transport of melanosomes in melanocytes and lytic granules in cytotoxic T-lymphocytes. Rab27 acts through organelle-specific effector proteins, such as granuphilin in pancreatic beta cells and melanophilin in melanocytes. The Rab27 and effector complex then interacts with proteins that are essential for membrane transport and fusion, such as syntaxin 1a and Munc18-1 for granuphilin and myosin Va for melanophilin. Genome information suggests that other putative Rab27 effector proteins, tentatively termed as exophilins or Slp/Slac2, are predicted to exist because these proteins share the conserved N-terminal Rab27-binding domain and show Rab27-binding activity in vitro or when overexpressed in cell lines. These findings suggest that the Rab27 subfamily regulates various exocytotic pathways using multiple organelle-specific effector proteins.

Melanosome Morphologies in Murine Models of Hermansky–Pudlak Syndrome Reflect Blocks in Organelle Development

Hermansky-Pudlak syndrome is an autosomal recessive disease characterized by pigment dilution and prolonged bleeding time. At least 15 mutant mouse strains have been classified as models of Hermansky-Pudlak syndrome. Some of the genes are implicated in intracellular vesicle trafficking: budding, targeting, and secretion. Many of the Hermansky-Pudlak syndrome genes remain uncharacterized and their functions are unknown. Clues to the functions of these genes can be found by analyzing the physiologic and cellular phenotypes. Here we have examined the morphology of the melanosomes in the skin of 10 of the mutant mouse Hermansky-Pudlak syndrome strains by transmission electron microscopy. We demonstrate that the morphologies reflect inhibition of organelle maturation or transfer. The Hermansky-Pudlak syndrome strains are classified into morphologic groups characterized by the step at which melanosome biogenesis or transfer to keratinocytes is inhibited, with the cappuccino strain observed to be blocked at the earliest step and gunmetal blocked at the latest step. We show that all Hermansky-Pudlak syndrome mutant strains except gunmetal have an increase in unpigmented or hypopigmented immature melanosomal forms, leading to the hypopigmented coat colors seen in these strains. In contrast, the hypopigmentation seen in the gunmetal strain is due to the retention of melanosomes in melanocytes, and inefficient transfer into keratinocytes.

Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome.

Griscelli syndrome (GS) patients and the corresponding mouse model ashen exhibit defects mainly in two types of lysosome-related organelles, melanosomes in melanocytes and lytic granules in CTLs. This disease is caused by loss-of-function mutations in RAB27A, which encodes 1 of the 60 known Rab GTPases, critical regulators of vesicular transport. Here we present evidence that Rab27a function can be compensated by a closely related protein, Rab27b. Rab27b is expressed in platelets and other tissues but not in melanocytes or CTLs. Morphological and functional tests in platelets derived from ashen mice are all within normal limits. Both Rab27a and Rab27b are found associated with the limiting membrane of platelet-dense granules and to a lesser degree with alpha-granules. Ubiquitous transgenic expression of Rab27a or Rab27b rescues ashen coat color, and melanocytes derived from transgenic mice exhibit widespread peripheral distribution of melanosomes instead of the perinuclear clumping observed in ashen melanocytes. Finally, transient expression in ashen melanocytes of Rab27a or Rab27b, but not other Rab's, restores peripheral distribution of melanosomes. Our data suggest that Rab27b is functionally redundant with Rab27a and that the pathogenesis of GS is determined by the relative expression of Rab27a and Rab27b in specialized cell types.

A Family of Rab27-binding Proteins: MELANOPHILIN LINKS Rab27a AND MYOSIN Va FUNCTION IN MELANOSOME TRANSPORT

The Rab27a GTPase regulates diverse processes involving lysosome-related organelles, including melanosome motility in melanocytes, and lytic granule release in cytotoxic T lymphocytes. Toward an understanding of Rab27a function, we searched for proteins that interact with Rab27a(GTP) using the yeast two-hybrid system and identified JFC1/Slp1, a protein of unknown function. JFC1/Slp1 and related proteins, including melanophilin, contain a conserved amino-terminal domain similar to the Rab3a-binding domain of Rabphilin-3. We used several methods to demonstrate that this conserved amino-terminal domain is a Rab27-binding domain. We show that this domain interacts directly, and in a GTP-dependent manner with Rab27a. Furthermore, overexpression of this domain in melanocytes results in perinuclear clustering of melanosomes, suggesting that this region is sufficient for interaction with, and perturbation of function of, Rab27a in a physiological context. Thus, we identified a novel family of Rab27-binding proteins. We also show that melanophilin associates with Rab27a and myosin Va on melanosomes in melanocytes, and present evidence that a domain within the carboxyl-terminal region of melanophilin interacts with the carboxyl-terminal tail of the melanocyte-specific splice isoform of myosin Va. Thus, melanophilin can associate simultaneously with activated Rab27a and myosin Va via distinct regions, and serve as a linker between these proteins.