Volume Changes of Individual Melanosomes Measured by Scanning Force Microscopy

Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G‐protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three‐dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P<0.05, n=200). The average ellipsoidal volume was 0.14±0.01 (aggregated) and 0.17±0.01 μm³ (dispersed), a difference of 18%. The average major diameter was 810±20 and 880±20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non‐destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.     

The electric charge of pigment granules in pigment cells

Black pigment cells called melanophores change colour in response to environmental changes and have lately been studied as promising biosensors. To further elucidate the intracellular processes involved in the colour changes of these cells, and to find optimal biosensing principles, the electric charge of intracellular pigment granules, melanosomes, has been determined in vitro by electrophoresis. Melanosomes from the two extreme states in the cell colour change (aggregated and dispersed melanosomes) were measured. The charge was found to be -1.5 x 10(-16) and -1.7 x 10(-16) C, aggregated and dispersed melanosomes, respectively, without significant difference between the two conditions. This charge is of the same order of magnitude as the one of 1000 electrons. The origin of the melanosome charge, and the use of these findings in new biosensor principles, is discussed.     

Effect of colcemid on the centrosome and microtubules in dermal melanophores of Xenopus laevis larvae in vivo.

An electron microscopy study showed that in melanophores with dispersed and aggregated pigment the sensitivity of the centrosome and the stability of microtubules were different and depended on the colcemid concentration. The structure of the centrosome didn't change upon exposure to colcemid in dispersed melanophores. In aggregated melanophores, on exposure to 10(-6) M colcemid, the centrosome retained its structure; colcemid at 10(-5)-10(-3) M caused a dramatic collapse of the centrosome. Treatment of aggregated melanophores with colcemid resulted in the complete disassembly of the microtubules; though microtubules in dispersed melanophores appear to be colcemid resistant. Light microscopy studies indicated that in Xenopus melanophores with aggregated or dispersed pigment melanosomes didn't change their location after exposure to 10(-3)-10(-6) M colcemid. Subsequent incubation in colcemid-free medium revealed that the cells retained their ability to translocate melanosomes in response to hormone stimulation. Electron microscopy data revealed the inactivation of the centrosome as MTOC (microtubule-organizing center) in dispersed melanophores with melatonin substituted for MSH in the presence of colcemid. In contrast, with melanocyte-stimulating hormone (MSH) substituted for melatonin, we observed the activation of the centrosome in aggregated cells. We showed that in aggregated melanophores pigment movement proceeded in the complete absence of microtubules, suggesting the involvement of a microtubule-independent component in the hormone-induced melanosome dispersion. However, we observed abnormal aggregation along colcemid-resistent microtubules in dispersed melanophores, suggesting the involvement of not only stable but also labile microtubules in the centripetal movement of melanosomes. The results raise the intriguing questions about the mechanism of the hormone and colcemid action on the centrosome structure and microtubule network in melanophores with dispersed and aggregated pigment.     

Actin and Tubulin Arrays in Cultured Xenopus Melanophores Responding to Melatonin

Melatonin induces pigment granule aggregation in amphibian melanophores. In the studies reported here, we have used fluorescence microscopic techniques to test the hypothesis that such melatonin-induced pigment movement is correlated with alterations in either the actin or tubulin cytoskeletal patterns of cultured Xenopus melanophores. In general, the cytoplasmic domains of the cultured melanophores were flat and thin except in the perinuclear region (especially when the pigment was aggregated). The microtubules and microfilaments were usually found in the same focal plane; however, on occasion, microfilaments were closer to the substratum. Microtubules were arranged in arrays radiating from what are presumed to be cytocenters. A small percentage of the melanophores were very large, had actin-rich circular perimeters and did not respond as rapidly to melatonin treatment as did the other melanophores. Melanophores with either aggregated or dispersed melanosomes had low intensity rhodamine-phalloidin staining of actin filaments compared to nonpigmented cells, whereas the FITC anti-tubulin intensities were comparable in magnitude to that seen in nonpigmented cells. When cells were fixed prior to complete melatonin-induced pigment granule aggregation there was no abrupt diminution in either the tubulin or actin staining at the boundary between pigment granule-rich and pigment granule-poor cytoplasmic domains. Nor could the actin and tubulin patterns in cells with partially aggregated melanosomes be reliably distinguished from those in melanophores in which the melanosomes were either completely dispersed or completely aggregated. These data argue against the hypothesis that melatonin causes consistent large-scale rearrangements of tubulin and actin polymers as it induces pigment aggregation in Xenopus melanophores.     

Control of melanosome movement in intact and cultured melanophores in the bitterling, Acheilognathus lanceolatus

The melanophores in the dermis on scales in the bitterling, Acheilognathus lanceolatus were studies to obtain information about the control mechanism of aggregation and dispersion using intact, membrane-permeabilized and cultured cells. The cultured melanophores showed supersensitivity, namely, they responded to norepinephrine with much higher sensitivity than intact cells. The cultured melanophores failed to respond to high KCl. Melatonin aggregated and adenosine dispersed melanosomes within a cell. Digitonin permeabilized cells showed aggregation with Ca ions and dispersion by cyclic adenosine 3',5'-monophosphate (cAMP) in the presence of ATP. Movement of melanosomes was observed under the high magnification of light microscope and the tracks of each pigment granule were followed. The granules moved fast and linearly during aggregation, whereas they showed to-and-fro movement during dispersion.     

Identification of Microtubule-Organizing Centers in Interphase Melanophores of Xenopus Laevis Larvae In Vivo

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immuno-stained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

The effect of cytochalasin B on pigment dispersion and aggregation in perfused Xenopus laevis tailfin melanophores

A perfusion technique is described for the study of melanosome response in ventral tailfin melanophores of Xenopus laevis tadpoles. The melanosomes remain aggregated (punctate melanophores) in Ringer's. Theophylline (15 mM) and caffeine (30 mM) cause a reversible dispersion (stellate melanophores) of melanosomes which is partly blocked by cytochalasin B (10 μg/ml). When added with theophylline or caffeine to stellate cells, cytochalasin B causes a disrupted distribution of pigment granules, characterized by a melanosome free central region. C-AMP (20 mM) and dibutyryl c-AMP (1 mM) cause a reversible dispersion of melanosomes which is partly inhibited by cytochalasin. When cytochalasin plus a nucleotide are added to stellate cells, some show the disrupted distribution of melanosomes. Colchicine (5 mM) causes irreversible, while griseofulvin (0.2 mM) causes a slight, but reversible dispersion of melanosomes, and cytochalasin has little effect on these reactions. Perfused tailfin melanophores remain capable of responding to reversible reagents for at least 12 hours and are unresponsive to changes in illumination.     

A role for spectrin in dynactin-dependent melanosome transport in Xenopus laevis melanophores

The bi-directional movement of pigment granules in frog melanophores involves the microtubule-based motors cytoplasmic dynein, which is responsible for aggregation, and kinesin II and myosin V, which are required for dispersion of pigment. It was recently shown that dynactin acts as a link between dynein and kinesin II and melanosomes, but it is not fully understood how this is regulated and if more proteins are involved. Here, we suggest that spectrin, which is known to be associated with Golgi vesicles as well as synaptic vesicles in a number of cells, is of importance for melanosome movements in Xenopus laevis melanophores. Large amounts of spectrin were found on melanosomes isolated from both aggregated and dispersed melanophores. Spectrin and two components of the oligomeric dynactin complex, p150(glued) and Arp1/centractin, co-localized with melanosomes during aggregation and dispersion, and the proteins were found to interact as determined by co-immunoprecipitation. Spectrin has been suggested as an important link between cargoes and motor proteins in other cell types, and our new data indicate that spectrin has a role in the specialized melanosome transport processes in frog melanophores, in addition to a more general vesicle transport.     

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

Aggregation of melanosomes in melanophores is accompanied by the reorganization of microtubules and intermediate filaments

The structure of the cytoskeleton in cultured melanophores of the fish Gymnocorymbus ternetzi during aggregation of melanosomes was studied. It has been shown that the motion of pigment granules is accompanied by a reorganization of microtubules and intermediate filament systems. In melanophores with dispersed pigment granules, microtubules are wavy and form a loose network whilst intermediate filaments in such cells form a dense network around the dispersed melanosomes. During aggregation microtubules and intermediate filaments become radially oriented. It was also shown that the surface area of melanophores increased during aggregation.     

The movement of melanosomes in melanophore fragments obtained by laser microbeam irradiation

The melanophores of the teleost Gymnocorymbus ternetzi are filled with pigment granules, melanosomes, which in response to appropriate treatments, can disperse throughout the cytoplasm or form an aggregate in the cell center. Melanophores with the dispersed pigment were irradiated by a laser microbeam, focused on the cell center by the microscope objective. If the average energy of the microbeam was 6-7 microJ, either the center of the melanophore was damaged and a single ring-shaped fragment was formed, or the cell was broken into several fragments of smaller size. The fragments retained their ability to move the pigment granules. In ring-shaped fragments, after adrenaline treatment, the melanosomes formed a ring-shaped aggregate moving away from both outer and inner (irradiation-produced) margins of the fragment. The smaller fragments treated with adrenaline moved the pigment to their centers. Both small and ring-shaped fragments could aggregate melanosomes as soon as 5 minutes after irradiation.     

A Role for Spectrin in Dynactin‐dependent Melanosome Transport in Xenopus laevis Melanophores

The bi‐directional movement of pigment granules in frog melanophores involves the microtubule‐based motors cytoplasmic dynein, which is responsible for aggregation, and kinesin  II and myosin  V, which are required for dispersion of pigment. It was recently shown that dynactin acts as a link between dynein and kinesin  II and melanosomes, but it is not fully understood how this is regulated and if more proteins are involved. Here, we suggest that spectrin, which is known to be associated with Golgi vesicles as well as synaptic vesicles in a number of cells, is of importance for melanosome movements in Xenopus laevis melanophores. Large amounts of spectrin were found on melanosomes isolated from both aggregated and dispersed melanophores. Spectrin and two components of the oligomeric dynactin complex, p150^glued and Arp1/centractin, co‐localized with melanosomes during aggregation and dispersion, and the proteins were found to interact as determined by co‐immunoprecipitation. Spectrin has been suggested as an important link between cargoes and motor proteins in other cell types, and our new data indicate that spectrin has a role in the specialized melanosome transport processes in frog melanophores, in addition to a more general vesicle transport.     

Microtubule dynamics in fish melanophores

We have studied the dynamics of microtubules in black tetra (Gymnocorymbus ternetzi) melanophores to test the possible correlation of microtubule stability and intracellular particle transport. X- rhodamine-or caged fluorescein-conjugated tubulin were microinjected and visualized by fluorescence digital imaging using a cooled charge coupled device and videomicroscopy. Microtubule dynamics were evaluated by determining the time course of tubulin incorporation after pulse injection, by time lapse observation, and by quantitation of fluorescence redistribution after photobleaching and photoactivation. The time course experiments showed that the kinetics of incorporation of labeled tubulin into microtubules were similar for cells with aggregated or dispersed pigment with most microtubules becoming fully labeled within 15-20 min after injection. Quantitation by fluorescence redistribution after photobleaching and photoactivation confirmed that microtubule turnover was rapid in both states, t1/2 = 3.5 +/- 1.5 and 6.1 +/- 3.0 min for cells with aggregated and dispersed pigment, respectively. In addition, immunostaining with antibodies specific to posttranslationally modified alpha-tubulin, which is usually enriched in stable microtubules, showed that microtubules composed exclusively of detyrosinated tubulin were absent and microtubules containing acetylated tubulin were sparse. We conclude that the microtubules of melanophores are very dynamic, that their dynamic properties do not depend critically on the state of pigment distribution, and that their stabilization is not a prerequisite for intracellular transport.     

Chromatophoromas in a pine snake

Iridophoroma and melanophoroma were diagnosed in an adult male pine snake. Light microscopic examination of irregularly thickened white and black portions of abnormal scales demonstrated two distinctive populations of pigment-containing cells. Pigment cells within abnormal-appearing white scales had needle-shaped granules that were dark amber in color while black portions were composed of pigment cells typical of melanophores, with dark black, round granules. Both populations of cells showed junctional activity, and clusters of both neoplastic pigment cell types were found in adjoining areas of the epidermis. By electron microscopy, the pigment cell with amber-colored granules contained reflecting platelet profiles typical of iridophores while pigment cells with dark round granules contained melanosomes. At a junctional area between abnormal white and black scales, mosaic chromatophores containing reflecting platelet profiles and melanosomes were observed. At 1 1/2 years following initial diagnosis, the snake died and neoplastic iridophores were found at multiple visceral sites; there was no evidence of metastases of melanophores to any organ. The two pigment cell tumors are believed to have developed from either stem cells destined to become iridophores and melanophores or from prexisting iridophores and melanophores in the dermis.     

Unusual responses of cultured Fundulus heteroclitus melanophores to epinephrine and ions, paradox of K+ versus Na+

Fundulus heteroclitus melanophores were successfully cultured and maintained in culture for up to 2 months. Compared to other teleost melanophores in the fin, scale, or split fin preparation, the cultured melanophores show unusual responses to both epinephrine and ion solutions. First, they aggregated their melanosomes in response to concentrations of epinephrine as low as 10(-12) M. Second, the melanophores in a 6-day, or older, culture aggregated their melanosomes in response to both sodium and potassium ion solutions. This is in contrast to 4-day-old cultures (and reports of noncultured melanophores) where melanosomes are aggregated in response to potassium but dispersed in sodium ion solutions.     

Novel receptors for melatonin that mediate pigment dispersion are present in some melanophores of the pencil fish (Nannostomus)

1. Comparing the daytime and the night-time pigmentary patterns of the skin of the pencil fish, Nannostomus beckfordi, we noticed that specific regions of dark spots that were part of the night-time pattern became pale during the day.2. Microscopic observations revealed that melanosomes in the melanophores in those regions were aggregated during the day but became dispersed at night.3. These melanophores responded to melatonin by dispersal of melanosomes while the cells on other parts of the body responded to melatonin by aggregation of the pigment in the normal way.4. The melanophores that responded to melatonin by pigment dispersion responded normally to other hormones and neurotransmitters, as did those on other parts of the skin.5. The results indicate that, in addition to the known melatonin receptor that mediates the aggregation of melanosomes, there also exists an unusual receptor which mediates the dispersion of pigment in melanophores. We have tentatively designated this receptor the ‘beta-melatonin receptor’.     

Effect of the barring gene on eye pigmentation in the fowl

Pigment cells of the iris, pecten, retinal pigment epithelium, and choroid of the wild-type jungle fowl (JF) and the barred Plymouth rock (BPR) breeds of adult chickens were studied at both light and electron microscopic levels. BPR choroidal tissues had 2.8 times fewer melanophores than the JF choroid, and BPR melanophores also contained 2.4 times fewer melanosomes, which tended to clump together in variously sized clusters. The melanosomes were often irregular in shape, smaller in diameter, and less mature (stage III) than those granules in the JF. The retinal pigment epithelium of both JF and BPR breeds contained a single epithelial layer of columnar cells. Rod-shaped melanosomes were present in the more apical regions of this cell type in both breeds. Both JF and BPR irides contained a multilayered posterior pigmented epithelium of columnar shaped cells that were densely filled with large spherical granules. Intercellular spaces with interdigitating cytoplasmic projections were present between pigment cells of both breeds. The pecten melanophores of both breeds were dendritic with melanosomes that were larger and fewer in numbers than those pigment cells of the iris and choroid. Intercellular spaces were present between cells in both breeds, with numerous villous-like pigment cell extensions. Choroid melanophores contained very little, if any, acid phosphatase activity. Approximately one-half of the retinal pigment epithelial cells observed contained small amounts of diffuse acid phosphatase activity in both breeds. The iris and pecten melanophores of both breeds contained profuse acid phosphatase activity scattered throughout their cytoplasms. Sparse tyrosinase activity was seen in iris and pecten pigment cells, whereas no tyrosine activity was observed in choroid melanophores or in retinal pigment epithelial cells in the two breeds, indicating that little new melanogenesis occurs in adult pigmented eye tissues. The results show that the barring gene reduces the number and melanin content of the choroidal melanophores in homozygous male BPR chickens as compared to the wild-type JF chickens. Whether this gene prevents the initial migration of embryonic neural crest cells (future melanophores) to the choroid or whether some of the choroidal melanophores prematurely degenerate in the embryo of young birds is yet to be determined. If the latter is the case, this choroid system may serve as a model for a genetic hypomelanotic disease such as vitiligo.     

The morphology of cultured melanophores from tadpoles of Xenopus laevis: Scanning electron microscopical observations

Summary Tail-fin melanophores of tadpoles of Xenopus laevis (Daudin) in primary culture were examined scanning electron microscopically in the aggregated and in the dispersed state. After isolation, the melanophores are spherical, but within 24 h they develop thin filopodia for attachment to the substratum. Subsequently, cylinder-like as well as flat sheet-like processes are formed, which adhere to the substratum with terminal pseudopodia and filopodia. The processes of adjacent melanophores contact each other, thus forming an interconnecting network between the melanophores.In the aggregated state the central part of the melanophore is spherical and voluminous. Both the central part and the processes bear microvilli. In melanophores with dispersed melanosomes the central part is much flatter; the distal parts have a thickness that equals a monolayer of melanosomes. The surface of the cell bears only scarce microvilli.These features indicate that melanophores do not have a fixed shape and that pigment migration is accompanied by reciprocal volume transformation between the cell body and its processes.     

An ultrastructural study of the melanophages in the cerebrospinal fluid of the tadpoles of Xenopus

Melanin deposits in the brain ventricles of Xenopus tadpoles were studied with light and electron microscopy (TEM and SEM). They appeared to be aggregations of melanophages which accumulated free pigment granules excreted by ependymal cells into the cerebrospinal fluid. Whereas the meningeal melanophores contained oval melanosomes of various sizes, the melanosomes in the scavenger cells were all spherical, large (0.6–1.1 μm) and fairly uniform in size. Moreover, they were arranged in spherical groups which were never seen in the cytoplasm of the melanophores. The melanosomes within the cells were identical to the free melanosomes found in the cerebrospinal fluid and those which occurred within the ependymal cells in the young larva, suggesting a common origin from the egg cytoplasm. The number of the melanosomes in the melanophages increased with age. Fine cytoplasmic projections were involved in catching and engulfing the melanosomes. Some other features of the cytoplasm, e.g., large deposits of cell detritus, also indicated that the cells were macrophages. In the later stages, (48, 49) no projections were observed, but the cells were totally filled with melanosomes.     

Possible Involvement of Cone Opsins in Distinct Photoresponses of Intrinsically Photosensitive Dermal Chromatophores in Tilapia Oreochromis niloticus

Dermal specialized pigment cells (chromatophores) are thought to be one type of extraretinal photoreceptors responsible for a wide variety of sensory tasks, including adjusting body coloration. Unlike the well-studied image-forming function in retinal photoreceptors, direct evidence characterizing the mechanism of chromatophore photoresponses is less understood, particularly at the molecular and cellular levels. In the present study, cone opsin expression was detected in tilapia caudal fin where photosensitive chromatophores exist. Single-cell RT-PCR revealed co-existence of different cone opsins within melanophores and erythrophores. By stimulating cells with six wavelengths ranging from 380 to 580 nm, we found melanophores and erythrophores showed distinct photoresponses. After exposed to light, regardless of wavelength presentation, melanophores dispersed and maintained cell shape in an expansion stage by shuttling pigment granules. Conversely, erythrophores aggregated or dispersed pigment granules when exposed to short- or middle/long-wavelength light, respectively. These results suggest that diverse molecular mechanisms and light-detecting strategies may be employed by different types of tilapia chromatophores, which are instrumental in pigment pattern formation.