Innervation of iridic melanophores

Summary The nerve supply to the iridic melanophores of the rat was studied with the electron microscope. The adrenergic and cholinergic terminals were identified with the aid of 5-hydroxydopamine, which produces dense-cored 400–800 Å synaptic vesicles in adrenergic axon varicosities, whereas the synaptic vesicles of cholinergic axons remain empty. It was found that both adrenergic and cholinergic terminal axons come in close apposition (200–250 Å) with the melanophores. The appositions have the same appearance as synapses in peripheral tissues. It seems likely that the murine iridic melanophores have a double innervation, although its functional significance is obscure.This work has been supported by grants from Lunds Läkarsällskap, the Swedish Medical Research Council (Project no. B69-14X-2321-02 and B69-14X-712-04C) and NIH (06701-02).     

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.     

Regulation of pigmentation in zebrafish melanophores

In comparison with the molecular genetics of melanogenesis in mammals, the regulation of pigmentation in poikilothermic vertebrates is poorly understood. Mammals undergo morphological colour change under hormonal control, but strikingly, many lower vertebrates display a rapid physiological colour change in response to the same hormones. The recent provision of extensive genome sequencing data from teleost zebrafish, Danio rerio, provides the opportunity to define the genes and proteins mediating this physiological pigment response and characterise their function biologically. Here, we illustrate the background adaptation process in adults and larvae and describe a novel assay to visualize and directly quantify the rate of zebrafish melanophore pigment translocation in unprecedented detail. We demonstrate the resolution of this assay system; quantifying the zebrafish melanophore response to melanin-concentrating and melanocyte-stimulating hormones. Furthermore, we investigate the intracellular signalling downstream of hormone stimulation and the biomechanical processes involved in zebrafish pigment translocation, confirming the importance of cyclic adenosine monophosphate (cAMP) as a mediator of pigment translocation and finding intact microtubules are essential for both melanin dispersion and aggregation in zebrafish, but that microfilament disruption affects aggregation only. In conclusion, we propose these data establish the zebrafish as an experimental model for studying both physiological colour change and the molecular basis of pigment translocation.     

Biosensing of opioids using frog melanophores

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.     

Localization and organization of actin in melanophores

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.     

Actin microfilaments in melanophores of Fundulus heteroclitus

Summary In melanophores of Fundulus heteroclitus, hormone-stimulated melanosome aggregation is accompanied by cytoplasmic flow from the cellular processes to the perikaryon, and reversal of these events takes place upon hormone-induced melanosome dispersion. These cells contain parallel arrays of microtubules, the majority of which are located in the perikaryon and in cortical regions of the processes. Studies with heavy meromyosin binding demonstrated two types of actin filaments: 1) a decorated meshwork of filaments similar to those usually found in close association with plasma membranes, and 2) filaments decorated in a manner similar to that of stress fibers. There is an apparent increase in the amount of filaments during melanosome aggregation. These results are discussed in relation to intracellular movement.Supported, in part, by grants AM-5384 and AM-13724 from U.S.P.H.S., and grant 234046 from the Japanese Ministry of Education     

Smoothened activates Galphai-mediated signaling in frog melanophores

The 7-pass transmembrane protein Smoothened was investigated for its ability to act as a G-protein-coupled receptor in Xenopus laevis melanophores. A plasmid containing the human Smoothened cDNA insert was transfected into immortalized frog pigment cells. Cells expressing the protein showed a phenotype of persistent pigment aggregation, a hallmark of constitutive Galpha(i) activation. Smoothened-mediated pigment aggregation was reversed by treatment with pertussis toxin or by co-expression with dominant negative Galpha(i). The ability of melanophores to express functional Smoothened was also determined by its co-expression with the twelve-pass transmembrane protein, Patched. Patched blocked Smoothened-mediated melanosome aggregation in a dose-dependent manner, consistent with its physiological role as an inhibitor of Smoothened. That the reconstituted Patched-Smoothened receptor complex functions normally in pigment cells was demonstrated by co-transfection with the activating ligand, Sonic hedgehog, as well as by direct application of the recombinant Sonic hedgehog protein. Sonic hedgehog reversed Patched-mediated inhibition of Smoothened and induced pigment aggregation. The findings demonstrate that the human Sonic hedgehog receptor complex can be functionally reconstituted in melanophores and that it is capable of transmembrane signaling by utilizing endogenous Galpha(i).     

Proliferation in vitro of melanophores from Xenopus laevis

Melanophores of wild-type and periodic albino mutants of Xenopus laevis were successfully cultured in vitro. They proliferated in the presence of alpha-melanocyte-stimulating hormone (alpha-MSH or cyclic adenosine monophosphate (cAMP) at a doubling time of 8-10 days. These proliferating melanophores retained their phenotypes, ability to synthesize melanin, and melanin-dispersing response to MSH stimulation. Neither depigmentation nor selective cell death of periodic albino melanophores was observed for at least 4 months during the cultivation.     

Statistics of active transport in Xenopus melanophores cells

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 μm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 μm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.     

Role of calcium in melanosome aggregation withinLabeo melanophores

In isolated scale melanophores ofLabeo rohita the melanosome aggregating effect of K⁺ was inhibited in Ca²⁺ deprived medium. Moreover, the Ca²⁺-antagonists, verapamil and lanthanum inhibited the action of K⁺ in concentration dependent manner. The elevation of extracellular Ca²⁺ could compromise the verapamil induced inhibition in a concentration dependent manner. The cation Ca²⁺ appeared to be required only for K+ -induced aggregation and not melanosome aggregationper se, as in this fish adrenaline and melanin concentrating hormone effectively caused aggregation of melanosomes in Ca²⁺ free medium     

Multiscale trend analysis of microtubule transport in melanophores

Microtubule-based transport is critical for trafficking of organelles, organization of endomembranes, and mitosis. The driving force for microtubule-based transport is provided by microtubule motors, which move organelles specifically to the plus or minus ends of the microtubules. Motor proteins of opposite polarities are bound to the surface of the same cargo organelle. Transport of organelles along microtubules is discontinuous and involves transitions between movements to plus or minus ends or pauses. Parameters of the movement, such as velocity and length of runs, provide important information about the activity of microtubule motors, but measurement of these parameters is difficult and requires a sophisticated decomposition of the organelle movement trajectories into directional runs and pauses. The existing algorithms are based on establishing threshold values for the length and duration of runs and thus do not allow to distinguish between slow runs and pauses, making the analysis of the organelle transport incomplete. Here we describe a novel algorithm based on multiscale trend analysis for the decomposition of organelle trajectories into plus- or minus-end runs, and pauses. This algorithm is self-adapted to the characteristic durations and velocities of runs, and allows reliable separation of pauses from runs. We apply the proposed algorithm to compare regulation of microtubule transport in fish and Xenopus melanophores and show that the general mechanisms of regulation are similar in the two pigment cell types.     

Microtubular apparatus of melanophores: three dimensional organization

Microtubular organization in the melanophores of the angelfish, Pterophyllum scalare, has been studied by serial thin sectioning. The course of microtubules has been followed in sets of transverse serial sections taken from the centrosphere and a segment of a cell process, respectively. Microtubules arise from a prominent zone in the cell center, the central apparatus, which is composed of numerous, small, electron-dense aggregates. the number of these loosely distributed densities is highest in the center of the centrosphere, but they may also be found at its periphery. Microtubules insert into, or becomes part of, the dense material, or at least start in its vicinity. Dense aggregates may be separated from centrioles by several micrometers rather than only being closely associated with these organelles. At some distance from the organizing zone, most of the microtubules gradually assume a cortical arrangement, i.e., take a course within about 100 nm of the limiting membrane. Serial sections were used to trace all microtubules within a 6μm-long segment of a cell process. 94 percent of the microtubules observed in this segment run its entire length; it is conceivable, therefore that a considerable number of microtubules extend between the initiating site in the centrosphere and the outermost cell region. A three-dimensional model of the 6μm-long segment reveals that, despite changes in the cell process outline, microtubules maintain a strictly cortical arrangement which gives the impression of a microtubule "palisade" lining the cortex of the cell process. The features of the microtubular apparatus of angelfish melanophores are discussed in relation to factors controlling microtubule initiation and distribution.     

Regulation of organelle transport in melanophores by calcineurin

Previous studies have shown that pigment granule dispersion and aggregation in melanophores of the African cichlid, Tilapia mossambica, are regulated by protein phosphorylation and dephosphorylation, respectively (Rozdzial, M. M., and L. T. Haimo. 1986. Cell. 47:1061- 1070). The present studies suggest that calcineurin, a Ca2+/calmodulin- stimulated phosphatase, is the endogenous phosphatase that mediates pigment aggregation in melanophores. Aggregation, but not dispersion, is inhibited by okadaic acid at concentrations consistent with an inhibition of calcineurin activity. Inhibition of aggregation in melanophores that have been BAPTA loaded or treated with calmodulin antagonists implicate Ca2+ and calmodulin, respectively, in this process. Moreover, addition of calcineurin rescues aggregation in lysed melanophores which are otherwise incapable of aggregating pigment. Immunoblotting with an anticalcineurin IgG reveals that calcineurin is a component of the dermis, which contains the melanophores, and indirect immunofluorescence localizes calcineurin specifically to the melanophores. Finally, this antibody, which inhibits calcineurin's phosphatase activity (Tash, J. S., M. Krinks, J. Patel, R. L. Means, C. B. Klee, and A. R. Means. 1988. J. Cell Biol. 106:1625-1633), inhibits aggregation but has no effect on pigment granule dispersion. Together these studies indicate that retrograde transport of pigment granules to the melanophore cell center depends upon the participation of calcineurin.     

Molecular mechanisms of pigment transport in melanophores.

We present an overview of the research on intracellular transport in pigment cells, with emphasis on the most recent discoveries. Pigment cells of lower vertebrates have been traditionally used as a model for studies of intracellular transport mechanisms, because these cells transport pigment organelles to the center or to the periphery of the cell in a highly co-ordinated fashion. It is now well established that both aggregation and dispersion of pigment in melanophores require two elements of the cytoskeleton: microtubules and actin filaments. Melanosomes are moved along these cytoskeletal tracks by motor proteins. Recent studies have identified the motors responsible for pigment dispersion and aggregation in melanophores. We propose a model for the possible roles of the two cytoskeletal transport systems and how they might interact. We also discuss the putative mechanisms of regulation of pigment transport, especially phosphorylation. Last, we suggest areas of research that will receive attention in the future in order to elucidate the mechanisms of organelle transport.     

Calcium-dependent irreversible effect of ionophore A23187 on melanophores

The calcium ionophore, A231187, induces a Ca2+ -dependent movement (dispersion) of melanosomes within skin melanophores of the lizard, Anolis carolinensis, in vitro. The effects of A23187 are irreversible, since after repeated rinsing of the skins in the absence of the ionophore they will always darken in Ringer containing Ca2+ but will immediately lighten when transferred to Ca2+ -free Ringer. These results suggest that the ionophore is irreversibly localized to the melanophore membrane and that its melanosome-dispersing effect is continuously dependent upon extracellular calcium.