Melanosome Transport Research Articles

Further evidence for genotype-phenotype disparity in Griscelli syndrome

First described in 1978, Griscelli syndrome (GS) is a rare autosomal recessive disorder featuring pigmentary dilution of hair and skin.1 GS is caused by mutations in any of three genes encoding a tripartite protein complex essential for melanosome transport.2 GS1 (MIM214450) presents as hypomelanosis with primary neurological defects and is caused by mutations in MYO5A,3 and which also may be the same entity as Elejalde syndrome (MIM256710).4 This article is protected by copyright. All rights reserved.

Autophagy deficient melanocytes display a senescence associated secretory phenotype that includes oxidized lipid mediators

Autophagy is a recycling program which allows cells to adapt to metabolic needs and to stress. Defects in autophagy can affect metabolism, aging, proteostasis and inflammation. Autophagy pathway genes, including autophagy related 7 (Atg7), have been associated with the regulation of skin pigmentation, and autophagy defects disturb the biogenesis and transport of melanosomes in melanocytes as well as transfer and processing of melanin into keratinocytes. We have previously shown that mice whose melanocytes or keratinocytes lack Atg7 (and thus autophagy) as a result of specific gene knockout still retained functioning melanosome synthesis and transfer, and displayed only moderate reduction of pigmentation. In cell culture the Atg7 deficient melanocytes were prone to premature senescence and dysregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling. To elucidate the biochemical basis of this phenotype, we performed a study on global gene expression, protein secretion and phospholipid composition in Atg7 deficient versus Atg7 expressing melanocytes. In cell culture Atg7 deficient melanocytes showed a pro-inflammatory gene expression signature and secreted higher levels of C-X-C motif chemokine ligand -1,-2,-10 and −12 (Cxcl1, Cxcl2, Cxcl10, Cxcl12), which are implicated in the pathogenesis of pigmentary disorders and expressed higher amounts of matrix metalloproteinases -3 and -13 (Mmp3, Mmp13). The analysis of membrane phospholipid composition identified an increase in the arachidonic- to linoleic acid ratio in the autophagy deficient cells, as well as an increase in oxidized phospholipid species that act as danger associated molecular patterns (DAMPs). The secretion of inflammation related factors suggests that autophagy deficient melanocytes display a senescence associated secretory phenotype (SASP), and we propose oxidized lipid mediators as novel components of this SASP.

TRIF and MAVS pathway is essential to induce Rab27A and melanosome transportation by TLR3 agonist Poly(I:C) in human epidermal melanocytes

TRIF and MAVS pathway is essential to induce Rab27A and melanosome transportation by TLR3 agonist Poly(I:C) in human epidermal melanocytes

TLR3 agonist Poly (I:C) enhance Rab27A and melanosome transportation through TLR3 and MAVS pathway in human epidermal melanocytes

TLR3 agonist Poly (I:C) enhance Rab27A and melanosome transportation through TLR3 and MAVS pathway in human epidermal melanocytes

Melanogenesis and Melanosome Transportation Modulators from Medicinal Plants

Melanin is a pigment distributed in the human body. The excess accumulation of melanin contents on skin surface induces mottling and freckles which are unfavorable for maintaining good health and beauty. On the other hand, gray hair is caused by the decreasing of melanin production. Hence, to maintain appropriate melanin level in human body is desired strongly. Melanin is biosynthesized in melanosome in melanocyte by catalysis of tyrosinase, tyrosinase related protein-1 (TRP-1), and TRP-2 from L-tyrosine as a starting material, and the mature melanosome is transported to the keratinocytes or hair matrix cells by several kinds of related proteins, such as melanoregulin (Mreg), EBP50-PDZ interactor of 64kDa (EPI64), Rab27A, Slac-2a, etc. The keratinocytes or hair matrix cells accumulate melanosomes, and the skin or hair pigmentation is appeared by the differentiation of the cells. In order to modulate the pigmentation of the tissue in human body, a number of melanogenesis modulating compounds have been isolated and identified from medicinal plants as tyrosinase activity inhibitors and/or modulators on melanogenic enzymes expressions. However, there are a few reports evaluating the effects of compounds on melanosome transportation which influence the control of pigmentation. Therefore, search for the novel compounds modulating melanosome transportation as well as modulating melanognesis is recently desired. Keywords: Melanogenesis modulator, melanosome transportation modulator, medicinal plants, tyrosinase.

Cross-talk between interferon-gamma and interleukin-18 in melanogenesis

Skin is the largest organ in our body and strategically placed to provide a metabolically active biological barrier against a range of noxious stressors. A lot of inflammatory cytokines, which are increased after ultraviolet (UV) irradiation produced by keratinocytes or other immunocytes, are closely related to pigmentary changes, including interleukin-18 (IL-18) and interferon-γ (IFN-γ). In this study, the effect of cross-talk between IL-18 and IFN-γ on melanogenesis was investigated. Treatment with IL-18 resulted in a dose-dependent increase of melanogenesis, while IFN-γ made an opposite effect. This influence of IL-18 and IFN-γ was mediated by regulations of microphthalmia-associated transcription factor (MITF) and its downstream enzymatic cascade expressions. Furthermore, IFN-γ inhibited basal and IL-18-induced melanogenesis. IFN-γ increased signal transducer and activator of transcription-1 (STAT-1) phosphorylation to play its position in regulating melanin pigmentation, and its inhibitory effect could be prevented by Janus Kinase 1 (JAK 1) inhibitor. IFN-γ could inhibit melanogenesis by decreasing melanocyte dendrite formation. In addition, IFN-γ inhibited the expressions of Rab Pases to suppress the mature and transport of melanosomes. IL-18 could rapidly induce Akt and PTEN phosphorylation and p65 expression in B16F10 cells. When treatment with IL-18 and IFN-γ together, the phosphorylation level of Protein Kinase B (Akt) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) and expression of p65 NF-κB were inhibited, compared with treated with IL-18 only. Our studies indicated that IFN-γ could directly induce B16F10 cells apoptosis in vitro. Furthermore, we demonstrated that IFN-γ markedly up-regulated IL-18 binding protein (BP) production in normal human foreskin-derived epidermal keratinocytes in dose-dependent manner. UVB irradiation induced protease-activated receptor-2 (PAR-2) expression in NHEK, IFN-γ could inhibit this enhancement in a dose-dependent manner. These data suggest that IFN-γ plays a role in regulating inflammation- or UV-induced pigmentary changes, in direct/indirect manner.

Tropomyosin isoforms bias actin track selection by vertebrate myosin Va.

Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin-Tpm3.1 and actin-Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin-Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an "actin-Tpm code" that allows for spatial and temporal sorting of actomyosin function in the cell.

Inhibitory effect of 2-methyl-naphtho[1,2,3-de]quinolin-8-one on melanosome transport and skin pigmentation

Melanosomes are lysosome-related organelles with specialized capabilities of melanin synthesis and movement mediated by the Rab27a-Melanophilin-MyosinVa protein complex. In this study, we found that 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO) induced melanosome aggregation around the nucleus in melan-a melanocytes and in melan-a melanocytes/SP-1 keratinocyte co-cultures without inducing toxicity or changing the melanin content. Western blot and real-time PCR analyses showed that MNQO decreased expression of the Rab27a, Melanophilin and MyosinVa proteins and mRNAs, respectively, in melan-a melanocytes. In a reconstituted human epidermis model, treatment with 0.001% MNQO reduced skin pigmentation. Also, MNQO reduced skin pigmentation in brown guinea pigs induced by UVB irradiation. These results indicated that regulation of melanosome transport may serve as a good target for new skin depigmenting agents and MNQO itself could be a candidate.

Effect of Hoechunyangkyeok-San Extract on Melanogenesis

Forsythia fructus has been shown to have antioxidative, anti-inflammatory, antibacterial, anti-aging and whitening effects. Hoechunyangkyeok-san (Forsythia viridissima-prescription) is a traditional herbal medicine, which has been clinically used for treating febrile and inflammatory disorders. This work was carried out to investigate the skin whitening effects of Forsythia viridissima-prescription extract (a hydrolyzed extract of Hoechunyangkyeok-san: SID White HYC) on skin. The effects of SID White HYC were assessed the melanin contents in B161 melanoma cells and the pigmented equivalent with HMB45 and Fontana Masson staining in 3D skin model. Then, we examined the expression of major pigment enzymes regulating melanin synthesis and melanosome transport related proteins in B16F1 cells. SID White HYC significantly inhibited the melanin synthesis (56.7% and 30.6% inhibition at 100 μg/mL, intracellular and secreted, respectively) in B16F1 cells and 3D skin model. In addition, western blotting analysis showed that SID White HYC reduced the expression of melanin synthesis and melanosome transport related proteins in B16F1 cells. In clinical trials, the cream containing 0.05% SID White HYC showed skin depigmentation effect without any irritation. These results suggest that SID White HYC may be useful inhibition of melanogenesis and melanosome transport. Therefore, SID White HYC may have potential as a skin-whitening ingredient in cosmetics.

Interactions of allele E of the MC1R gene with FM and mutations in the MLPH gene cause the five-gray phenotype in the Anyi tile-like gray chicken

The Anyi tile-like gray chicken is a Chinese indigenous breed with a gray dilution phenotype, having gray feathers, comb, skin, shanks, and beak, which is valuable for genetic research on pigmentation. However, the genetic basis of the gray dilution phenotype remains unknown. The objective of this study was to investigate the genetic basis of the gray dilution phenotype in the Anyi tile-like gray chicken. We found that all Anyi tile-like gray chickens tested in this study carried at least one E allele, which is responsible for the appearance of black feathers, and some of them carried the FM allele, which is responsible for the black skin phenotype. A single nucleotide polymorphism (C.1909A>G) was identified within the melanophilin (MLPH) gene and was significantly associated with the gray dilution phenotype. Our findings suggest that the E and FM alleles act together to cause the development of the "five-black" phenotype (black feather, comb, skin, shank, and beak), whereas the MLPH mutation results in defective melanosome transport, leading to the development of the "five-gray" phenotype.

NINL and DZANK1 Co-function in Vesicle Transport and Are Essential for Photoreceptor Development in Zebrafish.

Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.

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.

Asymmetries in kinesin-2 and cytoplasmic dynein contributions to melanosome transport

The mechanisms involved in bidirectional transport along microtubules remain largely unknown. We explored the collective action of kinesin-2 and dynein motors during transport of melanosomes in Xenopus laevis melanophores. These motors are attached to organelles through accessory proteins establishing a complex molecular linker. We determined both the stiffness of this linker and the organelles speed and observed that these parameters depended on the organelle size and cargo direction. Our results suggest that melanosome transport is driven by two dissimilar teams: whereas dynein motors compete with kinesin-2 affecting the properties of plus-end directed organelles, kinesin-2 does not seem to play a similar role during minus-end transport.

Cdc42 and sec10 Are Required for Normal Retinal Development in Zebrafish.

To characterize the function and mechanisms of cdc42 and sec10 in eye development in zebrafish. Knockdown of zebrafish cdc42 and sec10 was carried out using antisense morpholino injection. The phenotype of morphants was characterized by histology, immunohistology, and transmission electron microscopy (TEM). To investigate a synergistic genetic interaction between cdc42 and sec10, we titrated suboptimal doses of cdc42 and sec10 morpholinos, and coinjected both morpholinos. To study trafficking, a melanosome transport assay was performed using epinephrine. Cdc42 and sec10 knockdown in zebrafish resulted in both abnormal eye development and increased retinal cell death. Cdc42 morphants had a relatively normal retinal structure, aside from the absence of most connecting cilia and outer segments, whereas in sec10 morphants, much of the outer nuclear layer, which is composed of the photoreceptor nuclei, was missing and RPE cell thickness was markedly irregular. Knockdown of cdc42 and sec10 also resulted in an intracellular transport defect affecting retrograde melanosome transport. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 act in the same pathway in retinal development. We propose a model whereby sec10 and cdc42 play a central role in development of the outer segment of the retinal photoreceptor cell by trafficking proteins necessary for ciliogenesis.

Quercetin derivatives regulate melanosome transportation via EPI64 inhibition and elongate the cell shape of B16 melanoma cells

4′-O-β-d-glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (3C4′GQ), first isolated from Helminthostachys zeylanica root extract, was synthesized as a compound that stimulates intracellular melanogenesis. 3-O-methylquercetin (3MQ) and 3,4′,7-O-trimethylquercetin (34′7TMQ) were synthesized as compounds that enhance extracellular melanin formation. The formation of dendrites and the expression of EBP50-PDZ interactor of 64kDa (EPI64) relating to melanin transportation were investigated using B16 melanoma cells treated with 3C4′GQ, 3MQ, or 34′7TMQ in order to understand the mechanism underlying the observed activities. The influence of 3C4′GQ on the increase of intracellular melanin contents enhanced the expression of EPI64, exhibited no dendrite elongation activity, and inhibited melanin transportation. On the other hand, the increase of extracellular melanin content by 3MQ and 34′7TMQ inhibited the expression of EPI64 and formed elongated cells to stimulate melanin transportation.

Rab1A regulates anterograde melanosome transport by recruiting kinesin-1 to melanosomes through interaction with SKIP

Melanosomes are lysosome-related organelles in melanocytes that are transported from the perinucleus to the cell periphery by coordination between bidirectional (anterograde and retrograde) microtubule-dependent transport and unidirectional actin-dependent transport. Although the molecular machineries that mediate retrograde transport and actin-dependent transport have already been identified, little is known about the anterograde transport complex on microtubules in mammalian cells. Here we discovered that small GTPase Rab1A on melanosomes recruits SKIP/PLEKHM2 as a Rab1A-specific effector and that Rab1A, SKIP, and a kinesin-1/(Kif5b+KLC2) motor form a transport complex that mediates anterograde melanosome transport in melanocytes. Interestingly, Arl8, Arf-like small GTPase that also interacts with SKIP, is specifically localized at lysosomes and regulates their anterograde transport in melanocytes. Our findings suggest that the anterograde microtubule-dependent transport of melanosomes and lysosomes are differently regulated by independent cargo receptors, i.e., Rab1A and Arl8, respectively, but that a SKIP–kinesin-1 mechanism is responsible for the transport of both.

Analysis of microRNA-203 function in CREB/MITF/RAB27a pathway: comparison between canine and human melanoma cells.

MicroRNA (miR)-203 is downregulated and acts as an anti-oncomir in melanoma cells. Here, using human and canine melanoma cells, we elucidated the effects of miR-203 on cyclic adenosine monophosphate response element binding protein (CREB)/microphthalmia-associated transcription factor (MITF)/RAB27a pathway, which is known to be important for the development and progression of human melanoma. In this study, we showed that miR-203 directly targeted CREB1 and regulated its downstream targets, MITF and RAB27a. miR-203 significantly suppressed the growth of human and canine melanoma cells and inhibited melanosome transport through the suppression of the signalling pathway. In conclusion, miR-203 was shown to be a common tumour-suppressive miRNA in human and canine melanoma and thus to play a crucial role in the biological mechanisms of melanoma development.

Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton.

Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F-actin cytoskeleton systems and resulted in β-tubulin degradation in an ubiquitin-proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F-actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F-actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport.

MicroRNA 340 is involved in UVB-induced dendrite formation through the regulation of RhoA expression in melanocytes.

The influence of UV irradiation on pigmentation is well established, but the molecular and cellular mechanisms controlling dendrite formation remain incompletely understood. MicroRNAs (miRNAs) are a class of small RNAs that participate in various cellular processes by suppressing the expression of target mRNAs. In this study, we investigated the expression of miRNAs in response to UVB irradiation using a microarray screen and then identified potential mRNA targets for differentially expressed miRNAs among the genes governing dendrite formation. We subsequently determined the ability of miRNA 340 (miR-340) to suppress the expression of RhoA, which is a predicted miR-340 target gene that regulates dendrite formation. The overexpression of miR-340 promoted dendrite formation and melanosome transport, and the downregulation of miR-340 inhibited UVB-induced dendrite formation and melanosome transport. Moreover, a luciferase reporter assay demonstrated direct targeting of RhoA by miR-340 in the immortalized human melanocyte cell line Pig1. In conclusion, this study has established an miRNA associated with UVB irradiation. The significant downregulation of RhoA protein and mRNA expression after UVB irradiation and the modulation of miR-340 expression suggest a key role for miR-340 in regulating UVB-induced dendrite formation and melanosome transport.

A role for Na+,K+-ATPase α1 in regulating Rab27a localisation on melanosomes.

The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.