Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo1

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long‐term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore‐fibroblast co‐culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast‐derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane‐enclosed melanosomes in a process that was upregulated by α‐melanocyte‐stimulating hormone (α‐MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane‐proteins seemed to be of importance, as the cluster‐like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long‐term colour changes and for studies of factors that affect pigmentation.     

Height Changes Associated with Pigment Aggregation in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Melanophores

Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgC1₂-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.     

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.     

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.     

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.     

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.     

Ultrastructure and distribution of chromatophores in the skin of the leopard gecko (Eublepharis macularius)

The aim of this study was to describe the ultrastructure and arrangement of pigment cells in the leopard gecko (Eublepharis macularius) skin to explain how wild‐type coloration is formed. The study also attempted to explain, on a morphological level, how skin colour changes occur. Samples of leopard gecko skin were collected from wild‐type coloration adult specimens. The morphology of pigmented cells was determined using light microscopy on haematoxylin and eosin (H&E) stained sections and in transmission electron microscopy. These studies indicate that skin of E. macularis contains xanthophores and melanophores but lacks iridophores and that this is probably related to nocturnal activity. The number and distribution of xanthophores and melanophores determines the skin colour and pigmentation pattern. The colour changes depend on the arrangement of characteristic protrusions of melanophores and the degree of filling them with melanosomes.     

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.     

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.     

Purification of Black Moor Goldfish melanophores and responses to epinephrine

Summary Two key modifications of the previously reported method for isolation of goldfish xanthophores allowed the isolation and establishment of primary cultures of terminally differentiated melanophores from the Black Moor goldfish (Carassius auratus). First, pretreatment with 10⁻⁴ M epinephrine causing aggregation of the melanosomes and collapse of the dendrites, prevents damage to the melanophores during tissue dissociation and melanophore isolation. Second, maintenance of these cells in culture was successful only when the culture medium was supplemented with fish serum. The purified melanophores attached, flattened, and were maintained in culture for up to 3 mo. Although the morphology of the cultured melanophores is less dendritic than their in vivo counterparts, the melanophores translocate melanosomes in a normal manner except that they exhibit enhanced sensitivity to epinephrine. This epinephrine-induced pigment aggregation, as well as the redispersion of pigment after the removal of epinephrine, can occur in the presence of ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid and absence of Ca²⁺. This work was supported by grant AM13724 from the National Institutes of Health, Bethesda, MD.     

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.     

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’.     

Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores

The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletal morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule-inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha-melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus-end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies.     

Enigmas of pterorhodin,a red melanosomal pigment of tree frogs

Melanosomes observed in dermal melanophores of adult leaf frogs contain a unique wine red pigment identified as pterorhodin, a pteridine dimer never before found in any vertebrate. This type of melanosome, almost twice as large as the typical eumelanin melanosome, contains a small electron dense core of eumelanin surrounded by a concentric fibrous mass of pterorhodin. Dermal melanophores of larval leaf frogs contain small eumelanin melanosomes that transform at metamorphosis through the gradual accumulation of pterorhodin on the eumelanin surface to form the compound melanosomes of adults. This process may be mediated by thyroxine. No explanation for the unique presence of pterorhodin in leaf frogs has yet surfaced. A variety of tree frog species from Australia and Papua New Guinea also possess pterorhodin and the large melanosome suggesting that tree frogs from the New World and those from Australia are closely related and may have been separated during continental drift. Several of the unsolved problems posed by the emergence of pterorhodin in a unique melanosome are discussed.     

Enigmas of Pterorhodin,a Red Melanosomal Pigment of Tree Frogs

Melanosomes observed in dermal melanophores of adult leaf frogs contain a unique wine red pigment identified as pterorhodin, a pteridine dimer never before found in any vertebrate. This type of melanosome, almost twice as large as the typical eumelanin melanosome, contains a small electron dense core of eumelanin surrounded by a concentric fibrous mass of pterorhodin. Dermal melanophores of larval leaf frogs contain small eumelanin melanosomes that transform at metamorphosis through the gradual accumulation of pterorhodin on the eumelanin surface to form the compound melanosomes of adults. This process may be mediated by thyroxine. No explanation for the unique presence of pterorhodin in leaf frogs has yet surfaced. A variety of tree frog species from Australia and Papua New Guinea also possess pterorhodin and the large melanosome suggesting that tree frogs from the New World and those from Australia are closely related and may have been separated during continental drift. Several of the unsolved problems posed by the emergence of pterorhodin in a unique melanosome are discussed.     

Localization of pigment cells in cultured frog skin

The pigmentation pattern of ventral skin of the frog Rana esculenta consists mainly of melanophores and iridophores, rather than the three pigment cells (xanthophores, iridophores, and melanophores) which form typical dermal chromatophore units in dorsal skin. The present study deals with the precise localization and identification of the types of pigment cells in relation to their position in the dermal tracts of uncultured or cultured frog skins. Iridophores were observed by dark-field microscopy; both melanophores and iridophores were observed by transmission electron microscopy. In uncultured skins, three levels were distinguished in the dermal tracts connecting the subcutaneous tissue to the upper dermis. Melanophores and iridophores were localized in the upper openings of the tracts directed towards the superficial dermis (level 1). The tracts themselves formed level 2 and contained melanophores and a few iridophores. The inner openings of the tracts made up level 3 in which mainly iridophores were present. These latter openings faced the subcutaneous tissue In cultured skins, such pigment-cell distribution remained unchanged, except at level 2 of the tracts, where pigment cells were statistically more numerous; among these, mosaic pigment cells were sometimes observed.     

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 microm3 (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.     

Effects of Odorants on Pigment Aggregation and cAMP in Fish Melanophores

Odor perception within olfactory neuroepithelium and pigment translocation within melanophores both seem to rely on a cAMP-based second messenger system. From studies on cultured frog melanophores, Lerner et al. (Proc. Natl. Acad. Sci. USA 85:261-264, 1988) suggested that some aspect of odor perception may be mediated by a nonspecific mechanism whose signal is transduced by a cAMP-based second messenger system. In the present study, odorants (β-ionone, benzylaldehyde, cineole, cinnamaldehyde, and octanol), which previously have been shown to stimulate formation of cAMP in the olfactory neuroepithelium, were investigated for possible pigment dispersing and cAMP-increasing effects. Pretreatment of fish melanophores with the adenylate cyclase activator forskolin (1 μM) resulted in an approximately 300% increase in cAMP and an almost complete blockage of noradrenaline-induced pigment aggregation. However, none of the tested odorants were able to increase the cAMP level and only cinnaldehyde and β-ionone were found to have any pigment dispersing activity.