Early Melanosomes Research Articles

Study on changing disciplinarian of beak colors in ducks and the regulation network based on transcriptome sequencing

Beak color in ducks is a primary characteristic of local breeds and genetic resources. Among them, black beaks, a rare packaging trait of high-quality duck products, have attracted much attention. In this study, Runzhou White Created ducks (black beak) and white-feathered Putian black ducks (yellow beak) were used to construct the F2 generation resource population to study the changing discipline of beak color combined with the beak color statistics of gray-beaked ducklings of Runzhou White Created ducks. Subsequently, transcriptome sequencing was performed to identify genetic markers related to beak color. To explore the rules of beak color change and its regulatory network, trends, and trend analysis and weighted gene co-expression network analysis（WGCNA）were performed. The screening results were verified by real-time quantitative polymerase chain reaction. A large difference was observed between the beak colors of birds from the F1 generation at 0 and 42 d of age. The F2 generation results show that nearly half of the black-beaked ducklings become green-beaked; the proportion of black spots for gray- and patterned-beaked ducklings increases with age, with most becoming green-beaked. Moreover, the beak color darkened from the first day, and the gray color value decreased significantly from the second day. Transcriptome sequencing indicated that TYR was differentially expressed between black and yellow beaks at 4 to 6 wk of age, and trend and WGCNA analyses showed that EDNRB signaling pathway genes and MITF were highly expressed in the first week, and TYR, TYRP1, and DCT were highly expressed at 4 to 6 wk of age. Therefore, there is melanin synthesis and deposition after hatching for gray- and patterned-beaked ducklings, while the yellow pigment might be deposited in the epidermis of beaks for black-beaked ducklings. The EDNRB signaling pathway is probably involved in early melanosome maturation and melanin formation in duck beaks, and genes such as TYR can maintain the black-beak phenotype.

Intermittent inhibition of FYVE finger-containing phosphoinositide kinase induces melanosome degradation in B16F10 melanoma cells.

Melanosomes are lysosome-related organelles that contain melanogenic factors and synthesize melanin as they mature. FYVE finger-containing phosphoinositide kinase (PIKfyve) regulates late endosome and lysosome morphology, vesicle trafficking, and autophagy. In melanocytes, PIKfyve inhibition has been reported to induce hypopigmentation due to impairments in the metabolism of early-stage melanosomes. Here, we report a new type of melanosome metabolism: post-PIKfyve inhibition, which was found during the characterization of the endosome/lysosome fluoroprobe GIF-2250. In B16F10 mouse melanoma cells, GIF-2250 highlighted vesicles positive for lysosomal-associated membrane protein 1 (lysosome marker) and other endosome/lysosome markers (CD63 and Rab7/9). When cells were continuously treated with PIKfyve inhibitors, intracellular vacuoles formed, while GIF-2250 fluorescence signals diminished and were diffusely distributed in the vacuoles. After removal of the PIKfyve inhibitors, the GIF-2250 signal intensity was restored, and some GIF-2250-positive vesicles wrapped the melanosomes, which spun at high speed. In addition, intermittent PIKfyve inhibition caused melanin diffusion in the vacuoles and possible leakage into the cytoplasmic compartments, and melanosome degradation was detected by a transmission electron microscope. Melanosome degradation was accompanied by decreased levels of melanin synthesis enzymes and increased levels of the autophagosome maker LC3BII, which is also associated with early melanosomes. However, the protein levels of p62, which is degraded during autophagy, were increased, suggesting an impairment in autophagy flux during intermittent PIKfyve inhibition. Moreover, the autophagy inhibitor 3-methyladenine does not affect these protein levels, suggesting that the melanosome degradation by the intermittent inhibition of PIKfyve is not mediated by canonical autophagy. In conclusion, disturbance of PIKfyve activity induces melanosome degradation in a canonical autophagy-independent manner.

First person – Christin Bissig

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Christin Bissig is first author on ‘The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation’, published in JCS. Christin conducted the research described in this article while a postdoc in Dr Guillaume van Niel's lab at the Institut Curie, Paris Sciences & Lettres Research University, France. She is now a postdoc in the lab of Andreas Mayer at Department of Biochemistry, University of Lausanne, Epalinges, Switzerland, investigating the molecular mechanisms of intracellular membrane trafficking.

The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.

ABCB6 Resides in Melanosomes and Regulates Early Steps of Melanogenesis Required for PMEL Amyloid Matrix Formation

Genetically inheritable pigmentation defects provide a unique opportunity to reveal the function of proteins contributing to melanogenesis. Dyschromatosis universalis hereditaria (DUH) is a rare pigmentary genodermatosis associated with mutations in the ABCB6 gene. Here we use optical and electron microscopy imaging combined with biochemical tools to investigate the localization and function of ABCB6 in pigment cells. We show that ABCB6 localizes to the membrane of early melanosomes and lysosomes of the human melanocytic cell line MNT-1. Depletion of ABCB6 by siRNA impaired PMEL amyloidogenesis in early melanosomes and induced aberrant accumulation of multilamellar aggregates in pigmented melanosomes. PMEL fibril formation and normal maturation of pigmented melanosomes could be restored by the overexpression of wild-type ABCB6 but not by variants containing an inactivating catalytic mutation (K629M) or the G579E DUH mutation. In line with the impairment of PMEL matrix formation in the absence of ABCB6, morphological analysis of the retinal pigment epithelium of ABCB6 knockout mice revealed a significant decrease of melanosome numbers. Our study extends the localization of ABCB6 to melanosomes, suggesting a potential link between the function of ABCB6 and the etiology of DUH to amyloid formation in pigment cells.

Cooperation of endothelin-1 signaling with melanosomes plays a role in developing and/or maintaining human skin hyperpigmentation.

ABSTRACTSkin hyperpigmentation is characterized by increased melanin synthesis and deposition that can cause significant psychosocial and psychological distress. Although several cytokine-receptor signaling cascades contribute to the formation of ultraviolet B-induced cutaneous hyperpigmentation, their possible involvement in other types of skin hyperpigmentation has never been clearly addressed. Since our continuous studies using skin specimens from more than 30 subjects with ethnic skin diversity emphasized a consistent augmentation in the expression of endothelin-1 (ET-1) and its receptor (Endothelin B receptor, ET-B) in hyperpigmented lesions, including senile lentigos (SLs), the precise function of ET-1 signaling was investigated in the present study. In line with previous studies, ET-1 significantly induced melanogenesis followed by increases in melanosome transport in melanocytes and in its transfer to keratinocytes while inhibition of ET-B function substantially depressed melanogenic ability in tissue-cultured SLs. Additionally, in agreement with a previous report that the formation of autophagosomes rather than melanosomes is stimulated according to starvation or defective melanosome production, ET-1 was found to remarkably augment the expression of components necessary for early melanosome formation, indicating its counteraction against autophagy-targeting melanosome degradation in melanocytes. Despite the lack of substantial impact of ET-1 on keratinocyte melanogenic functions, the expression of ET-1 was enhanced following melanosome uptake by keratinocytes. Taken together, our data suggest that ET-1 plays a substantial role in the development and/or maintenance of skin hyperpigmentation in reciprocal cooperation with increased melanosome incorporation.

Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome‐specific and proteolytically released protein

Melanosomes are organelles specialized for the production of melanin pigment and are specifically produced by melanocytic cells. More than 150 pigmentation-related genes have been identified, including glycoprotein nonmetastatic melanoma protein b (GPNMB). A recent proteomics analysis revealed that GPNMB is localized in melanosomes, and GPNMB is a membrane-bound glycoprotein that shows high homology with a well-known melanosomal structural protein, Pmel17/gp100. In this study, we show that GPNMB is expressed in melanocytes of normal human skin, as well as in human melanoma cells. GPNMB is heavily glycosylated and is enriched in mature (stage III and IV) melanosomes in contrast to MART-1 and Pmel17, which are abundant in early (stage I and II) melanosomes. MART-1 and Pmel17 play critical roles in the maturation of early melanosomes; thus, we speculate that GPNMB might be important in the functions of late melanosomes, possibly their transport and/or transfer to keratinocytes. We also demonstrate that a secreted form of GPNMB is released by ectodomain shedding from the largely Golgi-modified form of GPNMB and that the PKC and Ca(2+) intracellular signaling pathways regulate that shedding. We conclude that GPNMB is a melanosomal protein that is released by proteolytic ectodomain shedding and might be a useful and specific histological marker of melanocytic cells.

Electron tomography of early melanosomes: Implications for melanogenesis and the generation of fibrillar amyloid sheets

Melanosomes are lysosome-related organelles (LROs) in which melanins are synthesized and stored. Early stage melanosomes are characterized morphologically by intralumenal fibrils upon which melanins are deposited in later stages. The integral membrane protein Pmel17 is a component of the fibrils, can nucleate fibril formation in the absence of other pigment cell-specific proteins, and forms amyloid-like fibrils in vitro. Before fibril formation Pmel17 traffics through multivesicular endosomal compartments, but how these compartments participate in downstream events leading to fibril formation is not fully known. By using high-pressure freezing of MNT-1 melanoma cells and freeze substitution to optimize ultrastructural preservation followed by double tilt 3D electron tomography, we show that the amyloid-like fibrils begin to form in multivesicular compartments, where they radiate from the luminal side of intralumenal membrane vesicles. The fibrils in fully formed stage II premelanosomes organize into sheet-like arrays and exclude the remaining intralumenal vesicles, which are smaller and often in continuity with the limiting membrane. These observations indicate that premelanosome fibrils form in association with intralumenal endosomal membranes. We suggest that similar processes regulate amyloid formation in pathological models.

SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent Sodium-Calcium Exchange Activity That Regulates Human Epidermal Melanogenesis

A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.

Involvement of Dynein and Spectrin with Early Melanosome Transport and Melanosomal Protein Trafficking

Melanosomes are unique membrane-bound organelles specialized for the synthesis and distribution of melanin. Mechanisms involved in the trafficking of proteins to melanosomes and in the transport of mature pigmented melanosomes to the dendrites of melanocytic cells are being characterized, but details about those processes during early stages of melanosome maturation are not well understood. Early melanosomes must remain in the perinuclear area until critical components are assembled. In this study, we characterized the processing of two distinct melanosomal proteins, tyrosinase (TYR) and Pmel17, to elucidate protein processing in early or late steps of the secretory pathway, respectively, and to determine mechanisms underlying the subcellular localization and transport of early melanosomes. We used immunological, biochemical, and molecular approaches to demonstrate that the movement of early melanosomes in the perinuclear area depends primarily on microtubules but not on actin filaments. In contrast, the trafficking of TYR and Pmel17 depends on cytoplasmic dynein and its interaction with the spectrin/ankyrin system, which is involved with the sorting of cargo from the plasma membrane. These results provide important clues toward understanding the processes involved with early events in melanosome formation and transport.

Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes

Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms mu1A and mu1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of mu1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (mu1A or mu1B) showed two distinct distribution patterns that involved Pmel17, and only mu1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking mu1B expression. Finally, we established that expression of mu1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature.

MART-1 Is Required for the Function of the Melanosomal Matrix Protein PMEL17/GP100 and the Maturation of Melanosomes

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

Isolation of Melanosomes

The methods used to purify early and late melanosomes are detailed. These methods include the use of highly pigmented cells to maximize recovery and the use of various sucrose density gradients to separate melanosome fractions based on their density (which is determined in large part by the amount of dense melanin pigment that they contain). Early melanosomes lacking pigment must be further purified using free-flow electrophoresis.

Regulation of Tyrosinase Processing and Trafficking by Organellar pH and by Proteasome Activity

Pigmentation of the hair, skin, and eyes of mammals results from a number of melanocyte-specific proteins that are required for the biosynthesis of melanin. Those proteins comprise the structural and enzymatic components of melanosomes, the membrane-bound organelles in which melanin is synthesized and deposited. Tyrosinase (TYR) is absolutely required for melanogenesis, but other melanosomal proteins, such as TYRP1, DCT, and gp100, also play important roles in regulating mammalian pigmentation. However, pigmentation does not always correlate with the expression of TYR mRNA/protein, and thus its function is also regulated at the post-translational level. Thus, TYR does not necessarily exist in a catalytically active state, and its post-translational activation could be an important control point for regulating melanin synthesis. In this study, we used a multidisciplinary approach to examine the processing and sorting of TYR through the endoplasmic reticulum (ER), Golgi apparatus, coated vesicles, endosomes and early melanosomes because those organelles hold the key to understanding the trafficking of TYR to melanosomes and thus the regulation of melanogenesis. In pigmented cells, TYR is trafficked through those organelles rapidly, but in amelanotic cells, TYR is retained within the ER and is eventually degraded by proteasomes. We now show that TYR can be released from the ER in the presence of protonophore or proton pump inhibitors which increase the pH of intracellular organelles, after which TYR is transported correctly to the Golgi, and then to melanosomes via the endosomal sorting system. The expression of TYRP1, which facilitates TYR processing in the ER, is down-regulated in the amelanotic cells; this is analogous to a hypopigmentary disease known as oculocutaneous albinism type 3 and further impairs melanin production. The sum of these results shows that organellar pH, proteasome activity, and down-regulation of TYRP1 expression all contribute to the lack of pigmentation in TYR-positive amelanotic melanoma cells.

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

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

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

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

The Hermansky-Pudlak syndrome (HPS) protein is part of a high molecular weight complex involved in biogenesis of early melanosomes.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder in which oculocutaneous albinism, bleeding tendency and a ceroid-lipofuscin lysosomal storage disease result from defects of multiple cytoplasmic organelles: melanosomes, platelet dense granules and lysosomes. The HPS polypeptide, a 700 amino acid protein which is unrelated to any known proteins, is likely to be involved in the biogenesis of these different organelles. Here, we show that HPS is a non-glycosylated, non-membrane protein which is a component of two distinct high molecular weight complexes. In non-melanotic cells the HPS protein is contained almost entirely in an approximately 200 kDa complex that is widely distributed throughout the cytosol. In melanotic cells the HPS protein is partitioned between this cytosolic complex and a >500 kDa complex that appears to consist of the approximately 200 kDa complex in association with membranous components. Subcellular fractionation, immunofluorescence and immunoelectron microscopy studies indicate that the membrane-associated HPS complex of melanotic cells is associated with tubulovesicular structures, small non-coated vesicles, and nascent and early-stage melanosomes. These findings suggest that the HPS complex is involved in the biogenesis of early melanosomes.

Hermansky-Pudlak syndrome and pale ear: melanosome-making for the millennium.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid-lipofuscin lysosomal storage disease. These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles--melanosomes, platelet granules, and lysosomes--suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles. The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified. Functional studies indicate that the HPS protein is part of a high-molecular weight complex involved in the biogenesis of early melanosomes. Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles.

Clonal separation of mature melanocytes from premelanocytes in a diploid human cell strain: spontaneous and induced pigmentation of premelanocytes.

Strains of pigmented melanocytes can be derived reproducibly from normal human skin. Published procedures have been modified here to yield a strain, 'Nohm-1', comprising many unpigmented cells as well as cells with various degrees of pigmentation observable by light microscopy. The unpigmented cells contain early melanosomes (pigment organelles) and the specific enzyme tyrosinase. They are on average smaller and less dendritic than the pigmented cells. Nohm-1 cells show normal chromosomal banding patterns and normal proliferative behaviour, including senescence. They form no tumours in immunodeficient (nude) mice. Nohm-1 cells have been cloned and yield two distinct types of colony, depending on the progenitor cell. Well-pigmented melanocytes engender pure colonies of pigmented cells, but cells with little or no pigment can produce both unpigmented and pigmented progeny. Thus there is a separate cell type, or premelanocyte, which can differentiate spontaneously and stably in culture; this cell type includes both unpigmented and faintly pigmented cells. Usefully, most premelanocytes are viable after frozen storage, unlike well-pigmented melanocytes. Some components of the culture medium affect the proportion of pigmented cells in Nohm-1 cultures, and hence probably the ratio of mature melanocytes to premelanocytes. Rapid pigmentation can be induced artificially and simply, by using a medium with increased extracellular pH and tyrosine concentration.

Increased size of stage II and III melanosomes during PUVA therapy

Four different theories have been presented to show how early melanosomes are formed and at what stage in melanosome formation melanization starts. In the present investigation the perikaryon of melanocytes have been studied and the longitudinal and the transverse diameter of stage I-IV melanosomes estimated before and after PUVA therapy in 5 patients. An increase in the longitudinal diameter of stage II and III melanosomes was found, whereas no increase in stage I and IV melanosomes was detected. No increase in vesiculo-globular bodies was detected in stage I and II melanosomes after PUVA therapy, while in stage III melanosomes vesiculo-globular bodies appeared to have increased in number of PUVA therapy. It is concluded that during stimulated melanogenesis--as seen after PUVA therapy--an unknown mechanism is activated, causing increased amounts of structural proteins to be incorporated into each stage II melanosome. The vesiculo-globular bodies do not appear to take part in this process.