Amphibian Melanophores Research Articles

Molecular underpinnings of cytoskeletal cross-talk

Cross-talk between the microtubule and actin networks has come under intense scrutiny following the realization that it is crucial for numerous essential processes, ranging from cytokinesis to cell migration. It is becoming increasingly clear that proteins long-considered highly specific for one or the other cytoskeletal system do, in fact, make use of both filament types. How this functional duality of "shared proteins" has evolved and how their coadaptation enables cross-talk at the molecular level remain largely unknown. We previously discovered that the mammalian adaptor protein melanophilin of the actin-associated myosin motor is one such "shared protein," which also interacts with microtubules in vitro. In a hypothesis-driven in vitro and in silico approach, we turn to early and lower vertebrates and ask two fundamental questions. First, is the capability of interacting with microtubules and actin filaments unique to mammalian melanophilin or did it evolve over time? Second, what is the functional consequence of being able to interact with both filament types at the cellular level? We describe the emergence of a protein domain that confers the capability of interacting with both filament types onto melanophilin. Strikingly, our computational modeling demonstrates that the regulatory power of this domain on the microscopic scale alone is sufficient to recapitulate previously observed behavior of pigment organelles in amphibian melanophores. Collectively, our dissection provides a molecular framework for explaining the underpinnings of functional cross-talk and its potential to orchestrate the cell-wide redistribution of organelles on the cytoskeleton.

Possible application of melatonin treatment in human diseases of the biliary tract

Melatonin was discovered in 1958 by Aaron Lerner. Its name comes from the ability of melatonin to change the shape of amphibian melanophores from stellate to roundish. Starting from the 1980s, the role of melatonin in the regulation of mammalian circadian and seasonal clocks has been elucidated. Presently, several other effects have been identified in different organs. For example, the beneficial effects of melatonin in models of liver damage have been described. This review gives first a general background on experimental and clinical data on the use of melatonin in liver damage. The second part of the review focuses on the findings related to the role of melatonin in biliary functions, suggesting a possible use of melatonin therapy in human diseases of the biliary tree.

Identification of protein kinase C (PKC) isoforms in teleostean, amphibian and avian pigment cells.

The beta isoform of protein kinase C (PKC) has been described as the main isoform involved in the stimulation of melanogenesis in mammalian skin melanocytes. Little is known about PKC isoforms in non-mammalian pigment cells. In neopterigian fish (holostei and teleostei), PKC is associated with pigment granule aggregation within the pigment cells (skin lightening), whereas in elasmobranchs and tetrapods, the activation of PKC leads to pigment granule dispersion (skin darkening). In an attempt to a better understanding of this distinct functional behavior upon PKC activation, we decided to investigate the PKC isoforms expressed in pigment cell lines of teleost fish, amphibians and birds, using RT-PCR followed by cloning and sequencing. Our results demonstrate the presence of messenger RNA (mRNA) for the following PKC isoforms: beta 1, lambda and iota in GEM-81 cells (Carassius auratus erythrophoroma), beta 1, beta 2 and zeta in Xenopus laevis (amphibian) melanophores; beta 1 and lambda in Gallus gallus (chicken) primary melanocytes. Beta 1 PKC seems to be conserved throughout phylogeny, but the diversity of the other isoforms in the different groups may account for the functional differences after PKC activation, which are observed between teleost and tetrapod pigment cells.

18] Cultured amphibian melanophores: a model system to study melanopsin photobiology

Publisher Summary Melanopsin is expressed by scattered perikarya among the amacrine and ganglion cells of the primate retina. In amphibians, melanopsin is expressed in the retina, hypothalamus, iridial myocytes, and melanophores. Amphibian melanophore responses to light protect vital organs against ultraviolet irradiation and produce rapid color changes needed for camouflage responses and animal survival. From a technical standpoint, it is important to understand the regulation and expression of melanopsin to avoid misinterpreting the actions of other agents on amphibian melanophores studied in lighted environments. This chapter describes the Xenopus melanophore model system. It also describes methods for monitoring the effects of melanopsin gene overexpression on the photobiological response of cultured transgenic melanophores. As computerized image analysis techniques for easy and reliable automated measurement of pigment dispersion are developed, it is anticipated that assay technologies in the melanophore model system will become more sophisticated. Future transgenic studies are likely to use Xenopus tropicalis instead of Xenopus laevis as a melanophore source to avoid the complications of pseudotetraploidy.

Amphibian Melanophore Technology as a Functional Screen for Antagonists of G-Protein Coupled 7-Transmembrane Receptors.

Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-coupled receptors were established and evaluated, either as a primary screening utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors. Stably or transiently expressing melanophores responded selectively to respective effectors of the human calcium, cannabinoid, and neurokinin-1 receptors. Several selective cannabinoid receptor-binding inhibitors of known potency were characterized as agonists or antagonists of the human peripheral cannabinoid (CB(2)) receptor. The results were consistent with changes in cAMP content of hCB(2)-transfected human embryonic kidney (HEK) cells challenged with the same CB(2)-binding antagonists. A stable melanophore cell line expressing the human calcium receptor was used to screen a compound collection directly for functional antagonists, several of which were confirmed as antagonists in secondary screens by stimulating parathyroid hormone (PTH) secretion from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to toxic effects and validating melanophores as a primary screening modality. Also described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors. ()

Amphibian melanophores become photosensitive when treated with retinal

Culture Xenopus melanophores treated with all-trans retinal disperse their pigment granules when exposed to light, but fail to do so when maintained in retinoid-free medium. Two similar, but distinct, responses to retinal are described. One cellular cohort (fast cells) requires less than 4 h of retinal treatment to become photosensitive; the other (slow cells) requires up to 7 days of retinal treatment. Fast cells are usually, but not always, more sensitive to retinal, responding to concentrations as low as 1 nM retinal. Slow cells require 10–100 nM retinal before they become photosensitive. In both slow and fast cells, retinal isomers are more potent activators of photosensitivity than retinols. Retinoic acid is not effective when given alone. Pretreatment with retinoic acid, however, enhances the cellular sensitivity to retinal so that slow cells tend to assume the characteristics of fast cells. These findings demonstrate that melanophore responses to light in vitro vary as a function of culture conditions and that retinal can activate melanophore photosensitivity. © 1996 Wiley-Liss, Inc.

Melatonin receptors: Localization, molecular pharmacology and physiological significance

A pre-requisite to understanding the physiological mechanisms of action of melatonin is the identification of the target sites where the hormone acts. The radioligand 2-[125I]iodo-melatonin has been used extensively to localize binding sites in both the brain and peripheral tissues. In general these binding sites have been found to be high affinity, with Kd in the low picomolar range, and selective for structural analogues of melatonin. Also the affinity of these sites can generally be modulated by guanine nucleotides, consistent with the notion that they are putative G-protein coupled receptors. However, only a few studies have demonstrated that these putative receptors mediate biochemical and cellular responses. In the pars tuberalis (PT) and pars distalis (PD) of the pituitary, the amphibian melanophore and vertebrate retina, evidence indicates that melatonin acts to inhibit intracellular cyclic AMP through a G-protein coupled mechanism, demonstrating that this is a common signal transduction pathway for many melatonin receptors. However in the pars distalis the inhibition of calcium influx and membrane potential are also important mediators of melatonin effects. How many different forms or states of the melatonin receptor exist is unknown, but clearly the identification of the structure of the melatonin receptor(s) and its ability to interact with different G-proteins and signal transduction pathways are quintessential to our understanding of the physiological mechanisms of action of melatonin. In parallel the recent development of new melatonin analogues will greatly aid our understanding of the pharmacology of the melatonin receptor both in terms of the development of potent melatonin receptor antagonists and for the definition of receptor sub-types. The wide species and phylogenic diversity of melatonin binding sites in the brain has probably generated more questions than answers. Nevertheless the localization of melatonin receptors to the suprachiasmatic nucleus of the hypothalamus is at least consistent with circadian effects within the foetus and the adult. In contrast the PT of the pituitary presents an enigma in relation to the seasonal effects of melatonin. A model of how melatonin might mediate the timing of the circannual events through the PT is proposed.

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.

Melatonin-induced desensitization in amphibian melanophores.

Video-microscopic examination of pigment granule translocation in cultured amphibian melanophores provides continuous, real-time observation of cellular responses to hormonal and pharmacologic agents and is particularly useful for studying the mechanisms underlying melatonin-induced pigment aggregation. We have used such video-microscopic technology to show that pigment cells become refractory to prolonged melatonin treatment and that the speed at which the desensitized condition becomes evident is dependent upon the countervailing concentration of antagonistic hormone, alpha-melanocyte stimulating hormone (MSH). When melanophores were treated with 10 nM melatonin, desensitization occurred within 30 minutes in the presence of 5 ng/ml MSH, whereas five hours of melatonin treatment was required before the desensitized state was observed in the presence of 1 ng/ml MSH. The persistence of desensitization after melatonin removal depends upon the duration of initial melatonin exposure. When melanophores were treated with melatonin (10 nM) in the presence of 10 ng/ml MSH for two hours, the desensitized condition lasted less than 30 minutes; if the initial melatonin treatment was increased to four hours, however, the melanophores remained in the desensitized state for more than two hours after the melatonin was removed from the medium. During the course of these treatments, there was no substantial degradation of melatonin activity; i.e., a second population of melanophores responded normally to the melatonin-containing media overlying desensitized melanophores.

Homologies between the amino acid sequences of some vertebrate peptide hormones and peptides isolated from invertebrate sources

1. 1. The 4K-prothoracicotropic hormone (PTTH) or bombyxin and the melanization-reddish coloration hormone of the silkworm Bombyx mori resemble insulin and insulin-like growth factors. 2. 2. The family of adipokinetic/red pigment concentrating hormones has some similarity with glucagon. 3. 3. Members of the FMRFamide family are found in vertebrates as well as an invertebrates. 4. 4. In Locusta, a molecule immunologically and biologically related to amphibian melanophore stimulating hormone has been partially characterized. 5. 5. Enkephalins and enkephalin-related peptides occur in insects and other invertebrates. 6. 6. Peptides belonging to the tachykinin family have been isolated from molluscan ( Octopus) salivary glands and from insect nervous tissue ( Locusta migratoria). 7. 7. Invertebrate arginine-vasotocin homologs have been isolated from an insect ( Locusta migratoria) and from a mollusc ( Conus). 8. 8. In Leucophaea, Locusta and Drosophila, peptides resembling those of the vertebrate gastrin/cholecystokinin family have been identified. 9. 9. As the number of different neuro-/gut peptides with possible function(s) as hormone, neurotransmitter or neuromodulator is now estimated to be of the order of a few hundred, more similarities will probably show up in the near future.

Comparison of structural requirements of α-MSH and ACTH for inducing excessive grooming and pigment dispersion

α-MSH and ACTH-like peptides are known to play an important role in the adaptation of many vertebrates to a new environment. These peptides induce pigment dispersion in amphibian melanophores through a receptor-mediated mechanism. In this study we compared the structural requirements of these peptides for melanotropic activity on Xenopus laevis melanophores with those for inducing excessive grooming in the rat. With the exception of ACTH 1–24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle 4,-D-Phe 7]-α-MSH is extremely active in both assays. Weak agonists such as [Leu 9]-α-MSH did not possess antagonistic properties either in the melanophore assay or in the excessive grooming test. The data suggest that the mechanism of action of α-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores.

Long-term cultivation of amphibian melanophores: In vitro ageing and spontaneous transformation to a continuous cell line

Pure melanophore populations isolated from the tail skin of the tadpole, Rana catesbeiana, were mass cultured for a period of 2–3 years. All cell lines of amphibian melanophores studied exhibited growth crisis (in vitro ageing) followed by spontaneous transformation to a continuous cell line, as shown by changes in growth characteristics in mass culture and in clone culture, by the appearance of the cells, and by measurements of cell volumes. Even after becoming a continuous cell line, amphibian melanophores continued to have a diploid chromosome number (2 n=26) in three of four cell lines examined. The chromosome mode in one cell line, however, changed to thirty. Measurement of melanin dispersion after the addition of α-melanocyte-stimulating hormone suggested that the mechanism for melanin dispersion in melanophores changed during in vitro ageing.

Lavender, a chick melanocyte mutant with defective melanosome translocation: a possible role for 10 nm filaments and microfilaments but not microtubules.

Lavender is a mutation of chick neural-crest-derived melanocytes showing dilute feather pigmentation. This defect, previously attributed to a lack of attenuation of dendrites, was found to be due to a defect in melanosome translocation. The mutant phenotype, of melanincongested perikarya and pigmentless dendrites is expressed both in vivo and in vitro. Studies with colcemid and cytochalasin B suggest that the avian melanocyte resembles a dispersing amphibian melanophore in its requirement for microfilaments but not microtubules. Ultrastructural analysis revealed a normal complement of intracellular filaments. Microtubules, however, are scarce. Intermediate (10 nm) filaments surround and are closely associated with intracellular organelles, while microfilaments interconnect all filaments and organelles. While-cell centrifugation at 300 g showed that 10 nm filaments stream behind and appear to attach to mobile membrane-bound organelles including the nucleus, lipid granules and mitochondria, as well as melanosomes. It is suggested that all intracellular filaments, especially microfilaments and intermediate filaments, interconnect forming a network responsible for organelle motility.

The Pharmacology of the Pineal Gland

Only recently have a sufficient number of publications been available to legitimize a review of the pharmacology of the mammalian pineal organ. Two decades ago Kitay & Altschule reviewed the world literature on pineal physiology, which comprises several thousand papers, and concluded only that removal of the pineal, or administration of pineal extracts, somehow affected pigmentation in lower vertebrates and gonadal function in mammals (1). As the studies described below demonstrate, much more information is now available concerning the pharmacology of the pineal. This review subdivides present knowledge into two areas: (a) the effects on mammals of administering pineal extracts or pure synthetic or natural pineal constituents and (b) the effects of drugs and hormones on the pineal itself. As might be anticipated, the bulk of studies cited in both categories deals with the pineal hormone, melatonin. Melatonin was first isolated from bovine pineal extracts in 1958 by Lerner and his colleagues (2), who used as a marker the capacity of the hormone to aggregate the pigment granules in amphibian melanophores around the cell nucleus. Five years later, Wurtman et al (3) showed that melatonin affected a physiological function in mammals, that is, the size and secretion of the ovary, and subsequent studies have demonstrated that melatonin administration also modifies the growth, composition, and functional activities of numerous other organs. Only recently an assay was developed that allows quantification of the melatonin in human urine (4). The concentrations of the compound vary with a characteristic daily rhythm, peaking at night. The pineal's apparent role as the sole or major source of melatonin, the presence of melatonin in urine, and the demonstration that physiologic effects follow a pinealectomy or the administration of melatomin seem to justify labeling it a pineal hormone. Melatonin synthesis and pineal biosynthetic activity are generally controlled by the sympathetic nerves of this organ (5,6). Therefore, it should not be surprising that drugs known to modify the synthesis, release, or metabolism of norepinephrine in peripheral organs also affect pineal function. Melatonin is itself a derivative of another biogenic amine, serotonin, whose metabolism and actions are also affected by numerous drugs. Indeed, the pineal has often provided an apt tool for examining monoaminergic mechanisms for pharmacologists not specifically concerned with its particular functional properties.

Effects of ACTH on melanophores and iridophores isolated from bullfrog tadpoles

Melanophores and iridophores were isolated from bullfrog tadpoles by the methods of trypsin digestion and Ficoll density-gradient centrifugation and cultured in vitro in collagen-coated plastic dishes. Both chromatophores attached to the dish and formed dendritic structures. The addition of alkali-treated ACTH to the culture medium induced the expansion of melanophores and contraction of iridophores. A possible role of this hormone in the behavior of isolated chromatophores is discussed. Many studies have been made of the mechanism of the MSH effect on amphibian melanophores ( Bitensky and Burstein, 1965; Goldman and Hadley, 1969; van de Veerdonk, 1969; Brouwer, 1970) and iridophores ( Bagnara, 1958; Hadley and Goldman, 1972). These studies were, however, carried out on chromatophores in vivo or in isolated skins, and it has remained uncertain whether MSH directly controlled these chromatophores without influence by tissue surrounding them.

The ultrastructural localization of DOPA- 3H in differentiating amphibian melanophores grown in vitro

Explants of trunk neural crest from axolotl ( Ambystoma mexicanum) embryos were grown for 7 days in hanging drop cultures. In the differentiating melanophores, the Golgi apparatus and rough-surfaced endoplasmic reticulum (RER) are prominent and well developed. The cytoplasm is further characterized by groups of free ribosomes and extensive arrays of polyribosomes, an irregular system of smooth endoplasmic reticulum, yolk platelets, lipochondria, mitochondria, microtubules, vacuoles, vesicles, and numerous pigment granules in various stages of development. Although some newly formed premelanosomes are found near the Golgi and RER, most have no special topographic relationship to either organelle. Seven-day-old cultures were treated with tritiated 3,4-dihydroxyphenylalanine (DOPA- 3H) and studied by means of electron microscopic radioautography. The specific localization of radioactivity in premelanosomes shows that this organelle is the site of melanogenesis in differentiating amphibian melanophores. The time at which the premelanosome is first able to utilize exogenous DOPA coincides with the time at which the developing granule comes to contain thickened fibrils with periodic substructure. The earlier unpigmented premelanosome containing a meshwork of thin fibrils and small vesicles does not incorporate isotope. During the genesis of the melanosome there is a morphologically distinct stage at which the premelanosome acquires the capacity for melanin synthesis and deposition.

Effect of adenosine 3′,5′-monophosphate on melanin dispersion in the shore crab Carcinus maenas (L)

The mode of action of the melanin-dispersing hormone (MDH) in the shore crab Carcinus maenas was studied. The dose-response relationship for this hormone obtained from the sinus gland was compared to the response of the melanophores to injected adenosine 3′,5′-monophosphate (cyclic AMP). In agreement with data reported on the effect of cyclic AMP on amphibian melanophores, millimolar concentrations of this nucleotide were required to cause melanin dispersion in the crab. Although the activity of adenyl cyclase in epidermal preparations of the shore crab was increased about 8-fold when fresh sinus gland extracts were added to the incubation medium, this stimulation does not appear to be related to the MDH activity of these extracts. These findings are discussed with reference to the concept that the action of MDH on the melanophore may be mediated by cyclic AMP.

Melanocyte stimulating activities of the [formula omitted]-nitrophenyl sulfenyl derivatives of adrenocorticotropin and [5-glutamine] -α-melanotropin

The o -nitrophenyl sulfenyl derivative of adrenocorticotropin in which the single tryptophan residue of the molecule is modified, is found to be fully active in stimulating amphibian melanophores. The melanophore stimulating activity of [5-glutamine] -α-melanotropin, a synthetic analog of α-melanotropin, is also found to be undiminished by chemical modification of the tryptophan residue.

Endocrinology of the amphibian pineal.

McCord and Allen (1917) found that extracts of mammalian pineal glands contain a potent contracting agent of larval amphibian melanophores. Lerner and his co-workers determined the chemical structure of this principle and named it melatonin. This agent contracts dermal melanophores at a concentration as low as 10−10 g/ml. Both intact and eyeless larval amphibians blanch when placed in the dark, and the melanophore contraction which causes this lightening response is abolished by pinealectomy. The amphibian pineal contains photoreceptive elements similar to those found in the vertebrate lateral eyes, and these elements are inhibited by light but are stimulated in its absence. There is evidence for the presence of both HIOMT and melatonin the amphibian pineal. It has been proposed that the body-blanching response results from a direct stimulation of the pineal under conditions of darkness leading to a release of melatonin into the general circulation which is then responsible for a direct contracting effect on dermal melanophores. The cytophysiological effects of melatonin mimic those that take place in the body-blanching response. Since no other hormone or pharmacological agent duplicates this response, this is strong evidence that melatonin is a hormone that normally regulates body blanching. Other evidence for the support of this hypothesis is presented. Cytological features of both normal and melatonin-induced lightening indicate that the effects of melatonin are at the effector cell level rather than at either the hypothalamus or the pituitary. An inhibition of MSH-release by melatonin is not involved. Melatonin plays a normal role in young larvae to regulate the lightening response that takes place in darkness (the primary chromatic response). Neither melatonin nor the pineal play a role in the later (secondary stage) adaptive background responses of amphibians. As McCord and Allen first noted, the pineal may contain other substances which may have other physiological roles in amphibians as well as other vertebrates. These have been little studied.

Origin of amphibian premelanosomes and their relation to microtubules.

AbstractIn amphibian melanophores, premelanosomes appear to be formed in fenestrated cisternae of the Golgi complex. The premelanosomes may develop within the cisternae or vesicles containing melanoprotein may bud from the cisternae to form premelanosomes. Some micrographs also demonstrate a close association between microtubules and either premelanosomes or melanosomes. The microtubules themselves may originate from electron dense particles in the centrosphere region of the cell.