Aggregation Of Melanophores Research Articles

To study the effect of photosensitizer on melanophore responses of blue gourami (Trichogaster trichopterus).

The present study has been carried out to evaluate the effect of a photosensitizer- Methoxsalen, on dietary supplementation, on melanophores/pigmentation and to evaluate the persistence of change in melanophores after discontinuing the feeding of photosensitizer supplemented feeds to labyrinth fish, blue gourami (Trichogaster trichopterus). The fishes were fed with four feeds (E1, E2, E3 and control containing Methoxsalen @ 20, 100, 500 and 0.0 mg/kg) in three replicates and each replicate have ten (10) fishes. Total ninety fishes (90) were employed for experimental feeds and thirty (30) kept in three aquaria for control. Fishes were fedad libitum 12:00 Noon on every day for 15 days and after one hour feeding, the fishes were exposed for half and hour in sunlight. Water was changed every day after exposure to sunlight. The water quality was congenial for ornamental fishes. After 15 days of feeding the fishes were given control feed in all the aquaria to study and visualize the persistency of the increased colour and/or changes in the melanosomes in terms of aggregation and dispersion of melanophores. Scales were removed and examined under compound microscope for melanosomes/melanophores and overall pigmentation. On addition of Methoxsalen the behavioral changes in melanophores was recorded and was found to be dose-dependent and the darkening of scale appears more with increased number of pigment granules. The fundings of the present study is very useful to understand changes brought in the behaviour of melanosomes in the melanophores on feeding with photosensitizer, through dietary supplementation, to render ornamental fishes to look paoer and/or darker. Therefore, this findings, that aggregation and dispersion phenomenon of melanophores, has direct impact relation with the market value of ornamental fishes. Key words: Ornamental fish, photosensitizer, Trichogaster trichopterus, methoxsalen, chromatophores.

Melanophores in the stripes of adult zebrafish do not have the nature to gather, but disperse when they have the space to move

Animal skin pattern is one of the good model systems used to study the mechanism of pattern formation. Molecular genetic studies with zebrafish have shown that pigment cells play a major role in the mechanism of stripe formation. Among the variety of cellular events that may be involved in the mechanism, aggregation of melanophores has been suggested as an important factor for pattern formation. However, only a few experimental studies detected the migration ability of melanophores in vivo. Here, we tried to determine whether melanophores really have the ability to aggregate in the skin of zebrafish. Melanophores in the adult stripes are packed densely and they rarely move. However, when the neighboring pigment cells are killed, they move and regenerate the stripe pattern, suggesting that melanophores retain the migration ability. To analyze the migration, we ablated a part of the melanophores by laser to give free space to the remaining cells; we then traced the migration. Contrary to our expectation, we found that melanophores repulsed one another and dispersed from the aggregated condition in the absence of xanthophores. Apparent aggregation may be forced by the stronger repulsive effect against the xanthophores, which excludes melanophores from the yellow stripe region.

Actin Dynamics Is Essential for Myosin-Based Transport of Membrane Organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

Phosphoinositide 3-kinase is involved in Xenopus and Labrus melanophore aggregation

Melanophores are pigmented cells capable of quick colour changes through coordinated transport of their intracellular pigment granules. We demonstrate the involvement of phosphoinositide 3-kinase (PI3-K) in Xenopus and Labrus aggregation by the use of the PI3-K inhibitor, LY-294002. In Xenopus, wortmannin-insensitive PI3-K was found to be essential for the aggregation, mitogen-activated protein kinase (MAPK) activation and tyrosine phosphorylation of a 280-kDa protein, and for the maintenance of low cyclic adenosine 3′:5′-monophosphate (cAMP) during the aggregated state. Pre-aggregated cells disperse completely to LY-294002 at 50–100 μM, involving a transient elevation in cAMP due to adenylate cyclase (AC) stimulation or to inhibition of cyclic nucleotide phosphodiesterase (PDE). The inactive analogue LY-303511 did not induce dispersion at the same concentrations. PDE4 and/or PDE2 was found to be involved in melanosome aggregation. The similar kinetics of LY-294002 and various PDE inhibitors indicates that the elevation of cAMP might be due to inhibition of PDE. In Labrus melanophores, LY-294002 had a less dramatic effect, probably due to less dependence on PDE in regulation of cAMP levels. In Xenopus aggregation, we suggest that melatonin stimulation of the Mel1c receptor via G βγ activates PI3-K that, directly or indirectly via MAPK, activates PDE.

Regulation of melanosome movement by MAP kinase.

Our objectives were to further characterize the signaling pathways in melatonin-induced aggregation in Xenopus melanophores, specifically to investigate a possible role of mitogen-activated protein kinase (MAPK). By Western blotting we found that melatonin activates MAPK, which precedes melanosome aggregation measured in a microplate reader. Activation of MAPK, tyrosine phosphorylation of a previously described 280-kDa protein, and melanosome aggregation are sensitive to PD98059, a selective inhibitor of MAPK kinase. The MAPK activation is also decreased by the adenylate cyclase stimulant forskolin. In summary, we found that MAPK is activated during melatonin-induced melanosome aggregation. Activation was decreased by an inhibitor of MAPK kinase, and by forskolin. In addition to inhibition of cyclic adenosine 3',5'-monophosphate (cAMP), reduction in protein kinase A activity (PKA), and activation of protein phosphatase 2A, we suggest that melatonin receptors activate the MAPK cascade and tyrosine phosphorylation of the 280-kDa protein. Although the cAMP/PKA signaling pathway is the most prominent, our data suggest that simultaneous activation of the MAPK cascade is of importance to obtain a completely aggregated state. This new regulatory mechanism of organelle transport by the MAPK cascade might be important in other eukaryotic cells.

Synthesis of new arylalkoxy amido derivatives as melatoninergic ligands

Amido derivatives 10– 18 of the corresponding oxyamines were synthesised as melatoninergic ligands by the reaction of hydroxyphtalimide with the halogeno derivatives or the corresponding alcohols using Mitsunobu reaction conditions. The affinity of the compounds for chicken brain melatonin receptors and recombinant human MT 1 and MT 2 receptors was evaluated using 2-[ 125I]-iodomelatonin as the radioligand. Overall, the introduction of an oxygen atom in the amido chain was not a favourable parameter as the compounds were less potent than the corresponding deoxy derivatives. However, nanomolar compounds were obtained with the arylethyloxy derivatives ( 13c (R′=nPr), chicken brain, hMT 1, hMT 2, K i values: 4.8, 3.86, 2.4 nM, respectively) and the 2,7-dimethoxynaphthalene derivatives ( 17c (R′=nPr), chicken brain, hMT 1, hMT 2, K i values: 0.04, 0.13, 0.1 nM, respectively). The functional activity of these compounds was evaluated by the aggregation of melanophores in Xenopus laevis tadpoles and the potency was related to the affinity of the molecules for melatonin receptors. The compounds were found to be full agonists and compound 17a was 20-fold more potent than melatonin in this bioassay.

Noradrenaline- and melatonin-mediated regulation of pigment aggregation in fish melanophores.

The effects of melatonin and noradrenaline (NA) on bi-directional melanosome transport were analysed in primary cultures of melanophores from the Atlantic cod. Both agents mediated rapid melanosome aggregation, and by using receptor antagonists, melatonin was found to bind to a melatonin receptor whereas NA binds to an alpha2-adrenoceptor. It has previously been stated that melatonin-mediated melanosome aggregation in Xenopus is coupled with tyrosine phosphorylation of a so far unidentified high molecular weight protein and we show that although acting through different receptors and through somewhat different downstream signalling events, tyrosine phosphorylation is of the utmost importance for melanosome aggregation mediated by both NA and melatonin in cod melanophores. Together with cyclic adenosine 3-phosphate-fluctuations, tyrosine phosphorylation functions as a switch signal for melanosome aggregation and dispersion in these cells.

Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Melanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R1 and MCH‐R2, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R1 and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R1 (SLC‐1), the radioligand was [125I]–[Tyr13]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [125I]–[D‐Phe13, Tyr19]‐MCH served as radioligand. The bioassay used for MCH‐R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R1 and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe13 in [Phe13, Tyr19]‐MCH was well tolerated by both MCH‐R1 and B16 MCH‐R, change of configuration to D‐Phe13 in [D‐Phe13, Tyr19]‐MCH or [D‐Phe13]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R1. By contrast, the D‐Phe13 residue increased the affinity of [D‐Phe13, Tyr19]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R1 in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R1.

Mechanism by which an elevation of extracellular glucide concentration induces pigment aggregation in medaka melanophores.

An increase in glucide concentration induces pigment aggregation in melanophores in the skin on scales isolated from the medaka, Oryzias latipes. In this study, hexoses (including the common D-isomers of glucose, galactose, fructose, and mannitol) were examined. Denervated melanophores were refractory to such stimuli. An alpha-adrenolytic agent, phentolamine, effectively blocked the responses of normally innervated melanophores. The pigment-aggregating action of glucide was inhibited by withdrawal of Ca(2+) and Mg(2+) ions from the medium. A specific blocker of voltage-dependent N-type Ca(2+) channels, the omega-conotoxin GVIA, also inhibited the glucide action. The conclusion derived is that an elevation of glucide levels acts to open Ca(2+) channels of presynaptic membranes of sympathetic postganglionic fibers, and the consequently released adrenergic transmitter acts on the effector cells to induce the aggregation of their pigmentary organelles.

Melanophore aggregation in strong static magnetic fields.

Contradicting results can be found in the literature on effects from magnetic exposure to pigment cells. We have studied the influence of strong, static, homogenous magnetic fields of 8 and 14 T on melanophore aggregation during exposure to the field. Melanophores, black pigment cells, in fish are large flat cells having intracellular black pigment granules. Due to large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes. This is especially true for modeling nerve cells, since melanophores share stem cells with axons. Twenty experiments on black tetra fish fins were carried out in the two magnetic flux densities. The same number of control experiments were carried out in the magnet with the magnetic field turned off. Several factors, such as the degree of maximal aggregation, speed of aggregation, and irregularity of the speed, were examined. The statistical analysis showed no significant field effects on the aggregation, with one exception: the irregularity in aggregation speed in the 8 T field, compared to control. The believed reorientation of the cytoskeleton (microtubules) in the field or the induced Lorentz force on moving pigment granules, did not affect the aggregation.

An increase in extracellular Ca(2+) concentration induces pigment aggregation in teleostean melanophores.

An increase in the concentration of Ca(2+) ions in the external medium ([Ca(2+)](o)) induced pigment aggregation in melanophores of three species of freshwater teleosts examined. Denervated melanophores were refractory to elevations of [Ca(2+)](o). The pigment-aggregating action was inhibited by the sympathetic blocking agents, phentolamine, prazosin and yohimbine. Bretylium, an agent known to block the release of the neurotransmitter, interfered with the response effectively. Ca(2+) blockers, such as Mn(2+), verapamil and gallopamil, also inhibited the response, possibly by inhibiting Ca(2+) entry into the presynaptic elements of melanosome-aggregating fibers. The conclusion is that the increase in [Ca(2+)](o) may induce membrane depolarization of presynaptic nervous elements around the melanophores, which open the voltage-dependent Ca(2+) channels there. The liberation of adrenergic neurotransmitter follows, which induces the aggregation of pigment in melanophores.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R.

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G(alpha)q coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

Synthesis of phenalene and acenaphthene derivatives as new conformationally restricted ligands for melatonin receptors.

Conformationally restricted phenalene and acenaphthene derivatives 5 were synthesized from phenalen-1-one and acenaphthen-1-one derivatives using the Horner-Emmons reaction. The amines were prepared through the corresponding isocyanates by the Curtius reaction on the acids or by the reduction of the nitriles. Amido derivatives (R(3) = Me, Et, n-Pr, c-Pr) were prepared by acylation of the amines with the appropriate anhydrides or acid chlorides or by the reductive acylation of the nitriles. The affinities of the compounds for melatonin binding sites were evaluated in vitro in binding assays using chicken brain melatonin and the human mt(1) and MT(2) receptors expressed in HEK-293 cells. The functionality of the compounds was determined by the potency to lighten the skin of Xenopus laevis tadpoles. Highly potent compounds were obtained. The data highlighted the role of the methoxy group located in the ortho position to the ethylamido chain as compounds with picomolar affinities such as 14c were obtained (chicken brain, hmt(1), hMT(2) K(i) values = 0.02, 0.008, 0.069 nM, respectively). Compound 14c was equipotent to the corresponding dimethoxy derivative 15c (chicken brain, hmt(1), hMT(2) K(i) values = 0.07, 0.016, 0.1 nM, respectively). On the other hand, the restricted conformation of the amido chain did not influence selectivity for the cloned hmt(1) and hMT(2) receptors. These compounds were also potent agonists of melanophore aggregation in X. laevis. 15a,c were several hundred fold more potent than melatonin (EC(50) = 0.025, 0.004 nM, respectively). Conformational studies indicated that the minimum energy folded conformation of the ethylamido chain could constitute the putative active form in the receptor site in agreement with previous results.

Dynein, dynactin, and kinesin II's interaction with microtubules is regulated during bidirectional organelle transport.

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

Is Ca 2+ the second messenger in the response to melatonin in cuckoo wrasse melanophores?

Pigment aggregation in melanophores of Labrus ossifagus is controlled by an α 2-adrenoceptor and is somehow modulated by melatonin. The signal transduction mechanisms seem to involve both an attenuation of cAMP and an increase in intracellular Ca 2+, inhibiting protein kinase A or activating a phosphatase, respectively. These effects result in dephosphorylation, which in turn induces aggregation. Various α 2-adrenoceptor agonists attenuate cAMP levels or increase the concentration of intracellular Ca 2+. Noradrenaline, for example, lowers cAMP but does not affect the calcium signal whereas B-HT 920, an α 2-adrenoceptor specific agonist, does not induce a cAMP decrease but does appear to induce an increase in intracellular Ca 2+. This later inference is drawn from experimens with BAPTA/AM, an intracellular calcium chelator, which counteracts the aggregation induced by B-HT 920. Interestingly, the very potent α 2-adrenoceptor agonist medetomidine apparently activates both signal transduction pathways, which could explain its high efficacy in producing aggregation. Melatonin itself does not cause pigment aggregation, but it potentiates noradrenaline-induced aggregation. It has been suggested that melatonin receptors and α 2-adrenoceptors follow the same signal transduction pathway, i.e. an attenuation of cAMP. In our experiments, melatonin did not reduce cAMP levels; instead it appears to increase Ca 2+ concentration, since melatonin-potentiated aggregation was inhibited by BAPTA/AM. Thus, aggregation amplified by melatonin is probably not mediated by a further decrease in cAMP, but by the same signal transduction mechanism as B-HT 920, i.e. an increase in Ca 2+. This further strengthens the suggestion that melatonin and B-HT 920 bind to the same site, but it is unclear if that particular site is on the melatonin receptor or the α 2-adrenoceptor.

Synthesis of β-Substituted Naphth-1-yl Ethylamido Derivatives as New Melatoninergic agonists

Naphthalene melatoninergic ligands with alkyl groups (Me, Et, Pr, Bz) in the β position of the ethylamido chain were synthesised. The affinity of the compounds for chicken brain melatonin receptors was evaluated using 2-[125I]-iodomelatonin as the radioligand. An increase in the affinity was observed with the β-methyl derivatives and the greatest increase was seen with the (−) enantiomers. The introduction of a 2- or 7-MeO group on the naphthalene ring and the lengthening (Et, Pr) of the alkylamido chain gave potent compounds such as (−)1h (Ki=24pM). The functional activity of these compounds was evaluated by the aggregation of melanophores in Xenopus laevis tadpoles. The potency to produce lightening of the skin of Xenopus laevis was related to the affinities values of the molecules at melatonin chicken brain receptors. The most potent ligands were found to be full agonists and compound 1h was 25 fold more potent than melatonin in this bioassay.

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.

The putative melatonin receptor antagonist GR128107 is a partial agonist on Xenopus laevis melanophores.

1. GR128107 (3-(1-acetyl-3-methyl-piperidine)-5-methoxyindole) has previously been reported to be a competitive melatonin receptor antagonist in blocking melatonin inhibition of [3H]-dopamine release from rabbit retina, a response mediated by the MT2 receptor subtype. 2. GR128107, like melatonin, induced a rapid (maximum response in 60-90 min) pigment aggregation in a clonal line of Xenopus laevis melanophores. GR128107 behaved as a partial agonist (pEC50 8.58+/-0.03, n=3) with an Emax of 0.83 (relative to melatonin, pEC50 10.09+/-0.03, n=3). 3. The concentration-response curve for pigment granule aggregation to both melatonin and GR128107 was displaced in a parallel, rightward manner by melatonin receptor antagonists with very similar potencies; estimated pKB RJ252 (against melatonin 4.60/against GR128107 4.54) < GR135533 (6.40/6.14) < Luzindole (6.45/6.49) < S20929 (6.58/6.65) < 4-P-PDOT (6.73/6.85). 4. Both melatonin- and GR128107-induced pigment granule aggregation was prevented by pretreatment of melanophores with pertussis toxin (10-1000 ng ml(-1)). 5. Prolonged pre-treatment of melanophores with melatonin desensitized the pigment aggregation response to GR128107. In desensitized cells, the maximal aggregation produced by GR128107 was only 0.27+/-0.01 (n=4) and the pEC50 was reduced (vehicle 8.57+/-0.12; melatonin pre-treated 7.84+/-0.09, n=4). The maximal response to melatonin in desensitized melanophores was unchanged but the pEC50 was reduced (vehicle 10.49+/-0.03; melatonin pre-treated 9.83+/-0.04, n=4). 6. These results demonstrate that GR128107 induces pigment granule aggregation in Xenopus melanophores by activating a cell membrane melatonin receptor coupled via a pertussis toxin-sensitive G-protein. 7. The partial agonist activity of GR128107 in melanophores may be apparent because of the very high density of melatonin receptors in these cells (Bmax 1223 fmol mg protein(-1)) compared to the low density of sites in rabbit retina (Bmax 3.1 fmol mg protein(-1)). This suggestion is supported by the finding that GR128107, like melatonin, acted as a full agonist and inhibited forskolin-stimulation of cyclic AMP accumulation in NIH-3T3 cells expressing a high density of human mt1 or MT2 receptors.

Effects of Light on Plasma Somatolactin Levels in Red Drum (Sciaenops ocellatus)

The effects of illumination on circulating somatolactin (SL) levels were studied in red drum sampled at various times during a 24-h light–dark cycle (12L:12D) and during a 24-h period of constant light. Plasma SL concentrations were low in red drum sampled during the light phase (0.6±0.05–1.1±0.2 ng/ml). The levels were significantly elevated during the early-dark phase (5.8±1.0 ng/ml) and declined during the late-dark phase (0.39±0.05 ng/ml). In contrast, plasma SL levels remained low (0.4±0.1–1.3±0.4 ng/ml) in fish sampled throughout a 24-h constant light period following a 1-week exposure to the light–dark cycle. Circulating SL levels were also significantly elevated during the early-dark phase in fish maintained under a reversed light–dark cycle. The role of the eyes in mediating the SL response to light was evaluated by comparing circulating SL levels in optic-tract sectioned and enucleated fish with those of intact (control) fish. Plasma SL concentrations were significantly higher in optic-tract sectioned and enucleated fish (6.5±0.9–13.8±1.5 ng/ml) than in control fish (0.6±0.05–3.9±0.7 ng/ml) during both the early-dark phase and the early-light phase of the 24-h light–dark cycle. The absence of significant changes in plasma SL levels between the light and dark phases in optic-tract sectioned and enucleated fish appears to be due to a loss of light perception in these fish. Moreover, intact and sham-operated red drum maintained in constant darkness had dramatically increased plasma SL levels (18.8±2.0–24.8±1.8 ng/ml). The present results are consistent with our earlier findings that plasma SL levels are elevated in fish kept in constant darkness and in a dark-background tank (reduction in overall light levels) during the light phase. Taken together, these results suggest that plasma SL levels are elevated in red drum in the absence of light and in response to low illumination. Interestingly, the integument of the fish became light during the dark phase of the light–dark cycle. In our earlier studies, the increase of plasma SL concentrations was associated with aggregation of melanophores, and direct effects of SL on melanophore aggregation were demonstrated. Overall, our studies with red drum suggest a possible role of elevated SL levels on melanophore aggregation during the dark phase of the 24-h day–night cycle.

Microtubule aster formation by dynein-dependent organelle transport.

The interplay between microtubules and the motor enzyme, cytoplasmic dynein, is essential for organisation of the cytoplasm, organelle transport, and cell division in eukaryotic cells. During mitosis, cytoplasmic dynein organises microtubules into two spindle pole asters, as well as the comparable multiple cytoplasmic asters induced by the microtubule-stabilising agent taxol. The mechanisms behind this cell cycle-regulated organisation are, however, not fully understood. We report here that the unidirectional dynein-dependent pigment organelle aggregation in taxol-treated melanophores from Atlantic cod, induces multiple microtubule asters. Usually, the pigment aggregates to a central pigment mass in the cell center, but pigment aggregation in taxol-treated cells induces formation of several peripheral pigment clusters that each localise to the center of a microtubule aster formation. When a cell with previously formed peripheral pigment clusters redisperse pigment, the asters disappear. Upon a subsequent reaggregation of the pigment, the aster formations reappear. The results indicate that the pigment aggregation process organises the microtubules into these formations. Immuno-electron microscopy of isolated pigment organelles indicates the presence of several dynein molecules on each pigment organelle, making it possible for each organelle to interact with several microtubules and thereby focusing microtubule minus ends. The possibility of unidirectional dynein-dependent organelle movement for organising microtubules into asters during cell division, and similarities in signal transduction between mitosis and pigment movement, are discussed.