Melanosomes In Melanocytes Research Articles

The regulation of platelet-dense granules by Rab27a in the ashen mouse, a model of Hermansky-Pudlak and Griscelli syndromes, is granule-specific and dependent on genetic background

AbstractThe ashen (ash) mouse, a model for Hermansky-Pudlak syndrome (HPS) and for a subset of patients with Griscelli syndrome, presents with hypopigmentation, prolonged bleeding times, and platelet storage pool deficiency due to a mutation which abrogates expression of the Rab27a protein. Platelets of mice with the ashen mutation on the C3H/HeSnJ inbred strain background have greatly reduced amounts of dense granule components such as serotonin and adenine nucleotides though near-normal numbers of dense granules as enumerated by the dense granule-specific fluorescent dye mepacrine. Thus, essentially normal numbers of platelet dense granules are produced but the granule interiors are abnormal. Collagen-mediated aggregation of mutant platelets is significantly depressed. No abnormalities in the concentrations or secretory rates of 2 other major platelet granules, lysosomes and alpha granules, were apparent. Similarly, no platelet ultrastructural alterations other than those involving dense granules were detected. Therefore, Rab27a regulates the synthesis and secretion of only one major platelet organelle, the dense granule. There were likewise no mutant effects on levels or secretion of lysosomal enzymes of several other tissues. Together with other recent analyses of the ashen mouse, these results suggest a close relationship between platelet dense granules, melanosomes of melanocytes and secretory lysosomes of cytotoxic T lymphocytes, all mediated by Rab27a. Surprisingly, the effects of the ashen mutation on platelet-dense granule components, platelet aggregation, and bleeding times were highly dependent on genetic background. This suggests that bleeding tendencies may likewise vary among patients with Griscelli syndrome and HPS with Rab27a mutations.

The leaden gene product is required with Rab27a to recruit myosin Va to melanosomes in melanocytes.

The function of lysosome-related organelles such as melanosomes in melanocytes, and lytic granules in cytotoxic T lymphocytes is disrupted in Griscelli syndrome and related diseases. Griscelli syndrome results from loss of function mutations in either the RAB27A (type 1 Griscelli syndrome) or MYO5A (type 2 Griscelli syndrome) genes. Melanocytes from Griscelli syndrome patients and respective murine models ashen (Rab27a mutant), dilute (myosin Va mutant), and leaden exhibit perinuclear clustering of melanosomes. Recent work suggests that Rab27a is required to recruit myosin Va to melanosomes, thereby tethering melanosomes to the peripheral actin network and promoting melanosome retention at the tips of melanocytic dendrites. Here, we characterize the function of the leaden gene product. We show that Rab27a, but not myosin Va, can be localized to melanosomes in leaden melanocytes, suggesting that the leaden gene product acts downstream of, or in parallel to, Rab27a in melanocytes to promote recruitment of myosin Va to melanosomes. We also observed reduced levels of myosin Va protein in leaden and ashen melanocytes, suggesting that myosin Va stability is influenced by the leaden and ashen gene products. In leaden cytotoxic T lymphocytes, we observed that lytic granules polarize towards the immunological synapse and kill target cells normally. However, in contrast to melanocytes, we found that neither the leaden gene product (melanophilin) nor myosin Va was detectable in cytotoxic T lymphocytes. These results suggest that Rab27a interacts with different classes of effector proteins in melanocytes and cytotoxic T lymphocytes.

Influence of alpha-melanocyte-stimulating hormone and ultraviolet radiation on the transfer of melanosomes to keratinocytes.

The epidermal melanin unit in human skin is composed of melanocytes and keratinocytes. Melanocytes, located in the basal layer of the epidermis, manufacture melanin-loaded organelles called melanosomes. Through their dendritic processes, melanocytes distribute melanosomes to neighboring keratinocytes, where their presence confers to the skin its characteristic color and photoprotective properties. In this study, we used murine melanocytes and keratinocytes alone and in coculture to characterize the processes involved in melanosome transfer. Ultraviolet (UV) radiation induced an accumulation of melanosomes in melanocytes, whereas treatment with a-melanocyte-stimulating hormone (MSH) induced exocytosis of melanosomes accompanied by ruffling of the melanocyte membrane. We found that keratinocytes phagocytose melanosomes and latex beads equally well and that this phagocytic process was increased by exposure of keratinocytes to UV radiation or to MSH. Coculture of melanocytes and keratinocytes resulted in an increase in MSH released to the medium. Gene array analysis of MSH-treated melanocytes showed up-regulation of many genes associated with exocytosis. In our studies, we never observed cytophagocytosis of melanosome-filled processes. This result, together with the other findings, suggests that a combination of signals that increase melanosome production and release by melanocytes and that stimulate phagocytosis by keratinocytes are the most relevant mechanisms involved in skin tanning.

Deletion in the OA1 gene in a family with congenital X linked nystagmus

AIMSTo elucidate the molecular genetic defect of X linked congenital nystagmus associated with macular hypoplasia in three white males of a three generation family with clear features of ocular albinism...

AP-3 mediates tyrosinase but not TRP-1 trafficking in human melanocytes.

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.

Rab27a regulates the peripheral distribution of melanosomes in melanocytes.

Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myosinVa colocalize on the cytoplasmic face of peripheral melanosomes in wild-type melanocytes. However, the amount of myosinVa in melanosomes from Rab27a-deficient ashen melanocytes is greatly reduced. These results, together with recent data implicating myosinVa in the peripheral capture of melanosomes, suggest that Rab27a is necessary for the recruitment of myosinVa, so allowing the peripheral retention of melanosomes in melanocytes.

Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene

BackgroundRab GTPases are regulators of intracellular membrane traffic. The Rab27 subfamily consists of Rab27a and Rab27b. Rab27a has been recently implicated in Griscelli Disease, a disease combining partial albinism with severe immunodeficiency. Rab27a plays a key role in the function of lysosomal-like organelles such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Little is known about Rab27b.ResultsThe human RAB27B gene is organised in six exons, spanning about 69 kb in the chromosome 18q21.1 region. Exon 1 is non-coding and is separated from the others by 49 kb of DNA and exon 6 contains a long 3' untranslated sequence (6.4 kb). The mouse Rab27b cDNA shows 95% identity with the human cDNA at the protein level and maps to mouse chromosome 18. The mouse mRNA was detected in stomach, large intestine, spleen and eye by RT-PCR, and in heart, brain, spleen and kidney by Northern blot. Transient over-expression of EGF-Rab27b fusion protein in cultured melanocytes revealed that Rab27b is associated with melanosomes, as observed for EGF-Rab27a.ConclusionsOur results indicate that the Rab27 subfamily of Ras-like GTPases is highly conserved in mammals. There is high degree of conservation in sequence and gene structure between RAB27A and RAB27B genes. Exogenous expression of Rab27b in melanocytes results in melanosomal association as observed for Rab27a, suggesting the two Rab27 proteins are functional homologues. As with RAB27A in Griscelli Disease, RAB27B may be also associated with human disease mapping to chromosome 18.

Colocalization of dynactin subunits P150Glued and P50 with melanosomes in normal human melanocytes.

Melanocytic dendrites consist of a central core of microtubules (MT) and a subcortical actin network. In previous reports we showed the presence of MT-associated motor proteins kinesin and cytoplasmic dynein on the melanosomal surface, forming a link with MT (Vancoillie et al. J Invest Dermatol 2000;114:421-429; Vancoillie et al. Br J Dermatol 2000;143:258-306). We could also demonstrate the association of kinectin, the kinesin receptor, with melanosomes. The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. Therefore, in this study, we investigated the in vitro expression of dynactin subunits P150Glued and P50 in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription-polymerase chain reaction and northern blot analysis. In an attempt to gain an insight into the subcellular localization of dynactin, immunofluorescence and immunoelectron microscopy (IEM) studies were performed. The two isoforms of P150Glued and P50 are expressed in all studied skin cells. Immunofluorescence staining shows punctate distributions for P150Glued and P50 in melanocytes. P150Glued shows a clear centrosomal staining and accentuation in the dendrite tips. P50 is also accentuated in the perinuclear area and dendrite tips. Immunofluorescence double-labeling with a melanosome marker showed apparent colocalization of both P150Glued and P50 with melanosomes. By IEM, P50 is detected on the surface of the majority of melanosomes in melanocytes. The colocalization of different subunits of the dynactin complex with melanosomes is consistent with the earlier finding of cytoplasmic dynein association with melanosomes and supports the hypothesis that this complex could form a link between cytoplasmic dynein and the melanosomal membrane.

Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome.

Griscelli syndrome (GS, MIM 214450), a rare, autosomal recessive disorder, results in pigmentary dilution of the skin and the hair, the presence of large clumps of pigment in hair shafts and an accumulation of melanosomes in melanocytes. Most patients also develop an uncontrolled T-lymphocyte and macrophage activation syndrome (known as haemophagocytic syndrome, HS), leading to death in the absence of bone-marrow transplantation. In contrast, early in life some GS patients show a severe neurological impairment without apparent immune abnormalities. We previously mapped the GS locus to chromosome 15q21 and found a mutation in a gene (MYO5A) encoding a molecular motor in two patients. Further linkage analysis suggested a second gene associated with GS was in the same chromosomal region. Homozygosity mapping in additional families narrowed the candidate region to a 3.1-cM interval between D15S1003 and D15S962. We detected mutations in RAB27A, which lies within this interval, in 16 patients with GS. Unlike MYO5A, the GTP-binding protein RAB27A appears to be involved in the control of the immune system, as all patients with RAB27A mutations, but none with the MYO5A mutation, developed HS. In addition, RAB27A-deficient T cells exhibited reduced cytotoxicity and cytolytic granule exocytosis, whereas MYO5A-defective T cells did not. RAB27A appears to be a key effector of cytotoxic granule exocytosis, a pathway essential for immune homeostasis.

Nevus depigmentosus: Clinical features and histopathologic characteristics in 67 patients

Background: Nevus depigmentosus is defined as a congenital nonprogressive hypopigmented macule or patch that is stable in its relative size and distribution throughout life. The pathogenesis and histopathologic characteristics of nevus depigmentosus is not yet fully established. Objective: The purpose of this study was to investigate the clinical and histopathologic characteristics of nevus depigmentosus as well as its pathogenesis. Methods: A clinical survey was done with 67 patients diagnosed as having nevus depigmentosus. Two skin biopsy specimens each were taken from 18 patients: one from the central part of the depigmented lesion and another from the border of the lesion, including peri-lesional normal skin. The sections were stained with hematoxylin-eosin, Fontana-Masson, and S-100 protein. Ultrastructural evaluation was also done to detect changes of the melanocytes. Results: The lesions were mostly present before 3 years of age (92.5%), but some lesions also appeared later in childhood (7.5%). The back and buttocks were the most commonly affected sites, followed by the chest and the abdomen, the face, the neck, and the arms. Forty patients (59.7%) had the isolated type of nevus depigmentosus and 27 patients (40.3%) had the segmental type. Histopathologic studies showed that the staining ability of Fontana-Masson in nevus depigmentosus lesions decreased compared with perilesional normal skin. However, there were no changes in the numbers of melanocytes identified as S-100-positive cells in the basal layer. Electronmicroscopic studies revealed a great reduction in the number of melanosomes in melanocytes and some membrane-bound aggregated melanosomes were observed in keratinocytes. Conclusion: The results of this study support the hypothesis that nevus depigmentosus is caused by the functional defects of melanocytes and the morphologic abnormalities of melanosomes.(J Am Acad Dermatol 1999;40:21-6.)

Molecular genetic dissection of mouse unconventional myosin-VA: head region mutations.

The mouse dilute (d) locus encodes unconventional myosin-VA (MyoVA). Mice carrying null alleles of dilute have a lightened coat color and die from a neurological disorder resembling ataxia and opisthotonus within three weeks of birth. Immunological and ultrastructural studies suggest that MyoVA is involved in the transport of melanosomes in melanocytes and smooth endoplasmic reticulum in cerebellar Purkinje cells. In studies described here, we have used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles that vary in their effects on coat color and the nervous system. Seven of these mutations mapped to the MyoVA motor domain and are reported here. Crystallographic modeling and mutant expression studies were used to predict how these mutations might affect motor domain function and to attempt to correlate these effects with the mutant phenotype.

Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-Va gene.

Griscelli disease (OMIM 214450) is a rare autosomal recessive disorder characterized by pigmentary dilution, variable cellular immunodeficiency and onset of acute phases of uncontrolled lymphocyte and macrophage activation, leading to death in the absence of bone-marrow transplantation. The pigmentary dilution is characterized by a diffuse skin pigmentation, silvery hair, large clumps of pigments in the hair shafts (Fig. 1) and an accumulation of melanosomes in melanocytes, with abnormal transfer of the melanin granules to the keratinocytes. Immunological abnormalities are characterized by absent delayed-type cutaneous hypersensitivity and an impaired natural-killer cell function. A similar disorder has been described in the dilute lethal mouse--which, however, differs by the occurrence of a severe neurological disorder. The dilute locus encodes myosin-Va, a member of the unconventional myosin family. Myosins bind actin and produce mechanical force through ATP hydrolysis. Some members of this family are thought to participate in organelle-transport machinery. Because of the phenotype resulting in the dilute mouse and because of their potential role in intracellular transport, unconventional myosin-encoding genes were regarded as candidate genes for Griscelli disease. Here we report that the Griscelli disease locus co-localizes on chromosome 15q21 with the myosin-Va gene, MYO5a, and that mutations of this gene occur in two patients with the disease. Griscelli disease is therefore a human equivalent of dilute expression in the mouse.

Topical skin depigmentation agents

Novel topical dermatologic or cosme-ceutical agents are needed to treat cutaneous hyperpigmentation, since most currently available products typically contain hydroquinone (HQ), which has significant disadvantages. Although most studies have found HQ therapies to be efficacious, this compound is highly toxic and mutagenic to mammalian cells. Skin pigmentation results from the enzymatic conversion of tyrosine into melanin within the melanosomes of melanocytes, followed by transfer of the melanosomes into the keratinocytes of the epidermis. In order to identify novel inhibitors of skin pigmentation based on inhibition of tyrosinase, the key enzyme responsible for initiating melanogenesis, agent screening methods have been developed using both melanocyte cell cultures and cell-free enzymatic assays. An example of an HQ analog that demonstrates the desired activity in these assays is methyl gentisate (MG), which was discovered as an inhibitor of de novo melanogenesis in cultured melanocytes, with significantly reduced cytotoxicity and lack of mutagenic potential compared to HQ. A strategy is summarized for discovering new compounds capable of inhibiting melanogenesis, such as MG, and developing them into dermatologic or cosmeceutical products. In addition, consideration is given to the major differences in product registration requirements in various global markets for topical skin-light-ening agents.

Mouse fibroblast expressing human tyrosinase with DHICA-oxidase activity produces predominantly pheomelanin deposit in lysosome.

The melanogenic gene-transfected cell system serves as a useful tool for the study of the symphonic relation between melanin synthesis and intracellular organelles such as melanosomes in melanocytes. We constructed melanin-producing mouse fibroblasts by transfection of human tyrosinase cDNA to investigate the intracellular changes caused by tyrosinase expression. DHICA-oxidase (5,6-dihydroxyindole-2-carboxylic acid oxidase) activity without TRP-1 (Tyrosinase Related Protein-1) expression in the cells suggested that human tyrosinase also possesses a DHICA-oxidase activities different from mouse tyrosinase. Electron microscopic observation indicated that melanin-deposit organelles have some lysosomal features. These properties of melanin-deposit organelles in tyrosinase expressing fibroblasts provide one evidence for the hypothesis that melanosome is the specialized lysosome in melanocytes.

Partial albinism with immunodeficiency (Griscelli syndrome)

Partial albinism with immunodeficiency is a rare and fatal immunologic disorder characterized by pigmentary dilution and variable cellular immunodeficiency. To define the phenotype, therapy, and outcome, we retrospectively analyzed seven consecutive patients. Primary abnormalities included a silvery-grayish sheen to the hair, large pigment agglomerations in hair shafts, and an abundance of mature melanosomes in melanocytes, with reduced pigmentation of adjacent keratinocytes. Clinical onset occurred between the ages of 4 months and 4 years and was characterized by accelerated phases (lymphohistiocytic infiltration of multiple organs, including the brain and the meninges), triggered by viral and bacterial infections. Characteristic laboratory features included pancytopenia, hypofibrinogenemia, hypertriglyceridemia, and hypoproteinemia. Consistent immunologic abnormalities were characterized by absent delayed-type cutaneous hypersensitivity and impaired natural killer cell function. Some patients had secondary hypogammaglobulinemia, impaired major histocompatibility complex-mediated cytotoxic effects, a decreased capacity of lymphocytes to trigger a mixed lymphocyte reaction, or various functional granulocytic abnormalities. The disease seems to be invariably lethal without bone marrow transplantation; the mean age at the time of death was 5 years. Bone marrow transplantation has been performed in three cases; two patients died in the immediate posttransplantation period of infectious complications, but one patient is cured after a follow-up of 5 years. We conclude that partial albinism with immunodeficiency (Griscelli syndrome) can be differentiated from Chédiak-Higashi syndrome by pathognomonic histologic features. One of the underlying immunologic defects may be a defective function of natural killer cells, predisposing the patient to virus-associated hemophagocytic syndrome or accelerated phases. The prognosis is very poor unless early bone marrow transplantation is carried out. (J P EDIATR 1994;125:886-95)

Cronkhite‐Canada Syndrome

This article describes the light and electron microscopic studies from a macule and the surrounding lightly hyperpigmented skin of a patient with the Cronkhite-Canada syndrome. Increased numbers of melanin granules in keratinocytes, increased numbers of melanosomes in melanocytes, and areas with increased numbers of melanocytes were found in the macular lesion. Skin from both the macule and surrounding area were also characterized by compact hyperkeratosis as well as perivascular inflammation and exocytosis not previously reported in these patients.

A syndrome associating partial albinism and immunodeficiency

Two unrelated patients with partial albinism, frequent pyogenic infections and acute episodes of fever, neutropenia and thrombocytopenia are described. Their pigmentary dilution was characterized by large clumps of pigments in the hair shafts and an accumulation of melanosomes in melanocytes. Melanocytes had few short dendritic expansions, and keratlnocytes were hypoplgmented. No or few Langerhans' cells were detected in skin by electron microscopy and ATP-ase reactions. This pigmentary dilution, different from all other human albinisms, resembles the unique defect of the mutant dilute (d-d) mouse. Despite the presence of an adequate number of T and B lymphocytes, the patients were hypogammaglobulinemic, deficient in antibody production and incapable of manifesting delayed skin hypersensitivity or of rejecting skin grafts. Their leukocytes did not stimulate normal lymphocytes and could not generate cytotoxic cells during mixed leukocyte reaction. T lymphocytes of one patient were unable to exert a helper effect on the maturation of B lymphocytes into immunoglobulin-containing cells following in vitro stimulation with pokeweed mitogen. This suggests that the humoral deficiency might be secondary to a defect of helper T lymphocytes. Granulocytes did not show any morphologic abnormality, and their bactericidal activity was only moderately reduced. An Increased number of polymorphonuclear leukocytes with polar distribution of Concanavaline A (Con A) receptors (capping) was found in one patient and her parents. The family histories suggest that this syndrome is transmitted as an autosomal recessive character.

Influence of Prostaglandins E1, E2, and Arachidonate on Melanosomes in Melanocytes and Keratinocytes of Anagen Hair Bulbs in Vitro

Anagen hair bulbs were collected and maintained in organ culture for periods up to 24 hr. Prostaglandin E1, E2 and arachidonate were introduced to the cultures and stimulated a series of cellular events characterized by peripheral orientation of microfilaments in the dendritic processes of melanocytes, complexing of melanosomes; and degradation of melanosomes within keratinocytes. These events were dependent on the concentration of prostaglandins. In addition, the most potent agent for these events was arachidonate. The action of arachidonate was hypothesized to occur through the production of endogenous prostaglandins since arachidonate was ineffective in the presence of inhibitors of prostaglandin synthetase.

Ultrastructure of Giant Pigment Granules (Macromelanosomes) in the Cutaneous Pigmented Macules of Neurofibromatosis

Electron microscopy was used to elucidate the nature of the giant granules in the light-brown, or cafe-au-lait, macules from 6 patients with neurofibromatosis. It was shown that these giant granules were surrounded by a membrane system similar to that of normal melanosomes in melanocytes and keratinocytes. These giant granules contained several subunits: (i) highly electron-dense amorphous material; (ii) electron-lucent globular bodies (400 Å in diameter), characteristic also of the normal melanosomes in heavily pigmented skin and hair; (iii) electron-dense globular bodies (400–500 Å in diameter) with fine grains inside; and (iv) fine granular subtances, or grains, of moderate to high electron density. Several subtypes were found, suggesting that a morphologic sequence may exist in the development of these granules in melanocytes. The giant granules were found also in keratinocytes as a single unit, indicating that they were apparently transfered from melanocytes to keratinocytes as a single unit after the maturation. Because of many similarities with normal melanosomes, we suggest that these giant granules be called macromelanosomes. In addition to these macromalanosomes small ellipsoidal melanosomes that were normal and small spherical granules melanosomes like those found in melanosomes were also found in the cafe-au-lait lesions.

Ultrastructure of the Human Toenail I. Proximal Nail Matrix

Electron microscopic examination of the proximal matrix of the human toenail revealed that the proximal end was branched into a few to several rootlets. It was composed of essentially the same dorsal, apical and ventral portions in which active melanocytes, Langerhans' cells and a Merkel cell were found. The basal matrix cells had finger-like cytoplasmic projections into the surrounding mesoderm. Bundles of fine fibrils (anchoring bundles) streamed down from the basal lamina, particularly from the basal lamina covering the finger-like projections. Numerous slender villi, developed from the peripheral cytoplasm, rightly interlocked the basal matrix cells. Cell-to-cell junctions became rather loose as the basal cells left the basal layer. Melanosomes of melanocytes were not well developed in Caucasian nail matrix, some were well developed in the Japanese nail and usually fully melanized in the Negro nail. Only one Merkel cell was encountered. It contained dense, membrane-surrounded secretory granules and was connected to the neighboring keratinocytes with desmosomes.