Fish Chromatophores--From Molecular Motors to Animal Behavior.

Chromatophores are pigment-bearing cells of lower vertebrates, including fish that cater for the ability of individual animals to shift body coloration and pattern. Color change provides dynamic camouflage and various kinds of communication. It is also a spectacular example of phenotypic plasticity, and of significant importance for adaptation and survival in novel environments. Through different cellular mechanisms, color change can occur within minutes or more slowly over weeks. Chromatophores have different pigment types and are located not only in the skin, but also in the eyes and internally. While morphological color change, including seasonal color change, has received a lot of interest from evolutionary biologists and behavioral ecologists, the more rapid physiological color change has been largely a research subject for cell physiologists. In this cross-disciplinary review, we have highlighted emerging trends in pigment cell research and identified unsolved problems for future research.

Mixture effects between different azoles and ß-naphthoflavone on the CYP1A biomarker in a fish cell line.

The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) - CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist ß-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2-4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48h, whereas exposure to the omeprazole for 48h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50=1.3-7.7µM), whereas omeprazole had no effect on this activity (IC50=72µM). Exposure to 10µM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10µM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24h at concentrations that are known to disassemble microtubules at 24h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules.

Hormonal regulation of colour change in eyes of a cryptic fish.

Colour change of the skin in lower vertebrates such as fish has been a subject of great scientific and public interest. However, colour change also takes place in eyes of fish and while an increasing amount of data indicates its importance in behaviour, very little is known about its regulation. Here, we report that both eye and skin coloration change in response to white to black background adaptation in live sand goby Pomatoschistus minutes, a bentic marine fish. Through in vitro experiments, we show that noradrenaline and melanocyte concentrating hormone (MCH) treatments cause aggregation of pigment organelles in the eye chromatophores. Daylight had no aggregating effect. Combining forskolin to elevate intracellular cyclic adenosine monophosphate (cAMP) with MCH resulted in complete pigment dispersal and darkening of the eyes, whereas combining prolactin, adrenocorticotrophic hormone (ACTH) or melanocyte stimulating hormone (α-MSH) with MCH resulted in more yellow and red eyes. ACTH and MSH also induced dispersal in the melanophores, resulting in overall darker eyes. By comparing analysis of eyes, skin and peritoneum, we conclude that the regulation pattern is similar between these different tissues in this species which is relevant for the cryptic life strategy of this species. With the exception of ACTH which resulted in most prominent melanophore pigment dispersal in the eyes, all other treatments provided similar results between tissue types. To our knowledge, this is the first study that has directly analysed hormonal regulation of physiological colour change in eyes of fish.

Acoustic detection of melanosome transport in Xenopus laevis melanophores.

Organelle transport studies are often performed using melanophores from lower vertebrates due to the ease of inducing movements of pigment granules (melanosomes) and visualizing them by optical microscopy. Here, we present a novel methodology to monitor melanosome translocation (which is a light-sensitive process) in the dark using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. This acoustic sensing method was used to study dispersion and aggregation of melanosomes in Xenopus laevis melanophores. Reversible sensor responses, correlated to optical reflectance measurements, were obtained by alternating addition and removal of melatonin (leading to melanosome aggregation) and melanocyte-stimulating hormone (MSH) (leading to melanosome dispersion). By confocal microscopy, it was shown that a vertical redistribution of melanosomes occurred during the dispersion/aggregation processes. Furthermore, the transport process was studied in the presence of cytoskeleton-perturbing agents disrupting either actin filaments (latrunculin) or microtubules (nocodazole). Taken together, these experiments suggest that the acoustic responses mainly originate from melanosome transport along actin filaments (located close to the cell membrane), as expected based on the penetration depth of the QCM-D technique. The results clearly indicate the potential of QCM-D for studies of intracellular transport processes in melanophores.

Interactions of pharmaceuticals and other xenobiotics on key detoxification mechanisms and cytoskeleton in Poeciliopsis lucida hepatocellular carcinoma, PLHC-1 cell line.

Fish are exposed to chemicals, including pharmaceuticals, in their natural habitat. This study focuses on effects of chemicals, including nine classes of pharmaceuticals, on key detoxification mechanisms in a fish liver cell-line (PLHC-1). Chemical interactions were investigated on efflux pumps, P-glycoprotein (Pgp) and multidrug resistance associated proteins (MRP1/MRP2), and on biotransformation enzymes, cytochrome P450 (CYP1A/CYP3A). Diclofenac and troleandomycin inhibited efflux activities, whereas ethinylestradiol activated efflux function. Exposure to troleandomycin and ß-naphthoflavone induced MRP2 mRNA levels, but no effects were seen on MRP1 or Pgp expressions. Inhibition of CYP1A activities were seen in cells exposed to α-naphthoflavone, ß-naphthoflavone, clotrimazole, nocodazole, ketoconazole, omeprazole, ethinylestradiol, lithocholic acid, rifampicin and troleandomycin. Exposure to fulvestrant, clotrimazole and nocodazole resulted in induction of CYP1A mRNA levels. Although, exposure to nocodazole resulted in disassembled microtubules. A CYP3A-like cDNA sequence was isolated from PLHC-1, but basal expression and activities were low and the gene was not responsive to prototypical CYP3A inducers. Exposure to ibuprofen, lithocholic acid and omeprazole resulted in fragmentation of microtubules. This study revealed multiple interactions on key detoxification systems, which illustrates the importance of study effects on regulation combined with functional studies to provide a better picture of the dynamics of the chemical defense system.

Rapid color change in fish and amphibians - function, regulation, and emerging applications.

Physiological color change is important for background matching, thermoregulation as well as signaling and is in vertebrates mediated by synchronous intracellular transport of pigmented organelles in chromatophores. We describe functions of and animal situations where color change occurs. A summary of endogenous and external factors that regulate this color change in fish and amphibians is provided, with special emphasis on extracellular stimuli. We describe not only color change in skin, but also highlight studies on color change that occurs using chromatophores in other areas such as iris and on the inside of the body. In addition, we discuss the growing field that applies melanophores and skin color in toxicology and as biosensors, and point out research areas with future potential.

Effects of Roundup and glyphosate formulations on intracellular transport, microtubules and actin filaments in Xenopus laevis melanophores.

Glyphosate containing herbicides, such as Roundup, are commonly used and generally considered to be safe. However, some toxic effects are found on amphibians in vivo and human and mouse cells in vitro. In this study the effects of Roundup, glyphosate, glyphosateisopropylamine and isopropylamine were studied on intracellular transport by measuring aggregation capacity in Xenopus laevis melanophores. The chemicals inhibited retrograde transport of melanosomes in the range of 0.5-5mM. Cellular morphology and localization of microtubules and actin filaments were affected as determined by immunocytochemistry. Both glyphosate and Roundup decreased pH in the media. Acidic pH inhibited melanosome transport and altered microtubule and actin morphology in the absence of chemicals, while transport inhibiting concentrations of glyphosate, Roundup and glyphosateisopropylamine disassembled both microtubules and actin filaments. At physiological pH the effects of Roundup decreased whereas glyphosate failed to inhibit transport. Physiological pH decreases glyphosate lipophilicity and its diffusion into the cytoplasm. The Roundup formulation contains surfactants, such as POEA (polyetylated tallow amine) that increases membrane permeability allowing cellular uptake at physiological pH. Our results show that the effects of glyphosate containing compounds are pH-dependent and that they inhibit intracellular transport through disassembly of the cytoskeleton possibly by interfering with intracellular Ca(2+)-balance.

New insights into melanosome transport in vertebrate pigment cells.

Pigment cells of lower vertebrates provide an excellent model to study organelle transport as they specialize in the translocation of pigment granules in response to defined chemical cues. This review will focus on the well-studied melanophore/melanocyte systems in fish, amphibians, and mammals. We will describe the roles of melanin, melanophores, and melanocytes in animals, current views on how the three motor proteins dynein, kinesin, and myosin-V are involved in melanosome transport along microtubules and actin filaments, and how signal transduction pathways regulate the activities of the motors to achieve aggregation and dispersion of melanosomes. We will also describe how melanosomes are transferred to surrounding skin cells in amphibians and mammals. Comparative studies have revealed that the ability of physiological color change is lost during evolution while the importance of morphological color change, mainly via transfer of pigment to surrounding skin cells, increases. In humans, pigment mainly has a role in protection against ultraviolet radiation, but also perhaps in the immune system.

Melanophores: a model system for neuronal transport and exocytosis?

Black pigment cells, melanophores, from lower vertebrates are specialized in bidirectional and coordinated translocation of pigment granules, melanosomes, in the cytoplasm. Melanophores develop from the neuronal crest and are most abundant in the dermal and epidermal layers of the skin, where the intracellular distribution of the pigment significantly influences the color of the animal. The transport of pigment is dependent on an intact cytoskeleton and motor proteins associated with cytoskeletal components. The easily cultured melanophores have proved to be excellent models for organelle transport because the intracellular movements of pigment can be visualized via light microscopy, and the granules move in response to defined chemical signals. The ease of achieving a combination of morphological and functional transport studies is the advantage of the melanophore system, and studies on pigment cells have revealed new components of the transport machinery, including molecular motors, their adapters, and transfer of vesicles to other cells. Many cellular components are transported with a combination of the actin- and microtubule-based transport systems, and, since all eukaryotic organisms rely on functional intracellular transport and an intact cytoskeleton, studies on melanophores are important for many aspects of cell biology, including axonal transport. In this review, we present an overview of the research on the pigment transport system and the potential use of pigment cells as a model system.

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.

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

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 alpha-melanocyte-stimulating hormone (alpha-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.

Spirobicyclo[2.2.2]octane derivatives: mimetics of baccatin III and paclitaxel (Taxol).

The formylated spirobyclic alcohol was computer modeled to be a mimetic of paclitaxel. In this model, the formyl group was used as a truncated paclitaxel side chain in order to reduce the computational work. Compound , carrying the paclitaxel side chain, was synthesized in six steps from optically active 1,3-diketone . Microtubule stabilization was not observed for , indicating that the model needs to be adjusted.

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.

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.

Regulatory control of both microtubule- and actin-dependent fish melanosome movement.

In fish melanophores, melanosomes can either aggregate around the cell centre or disperse uniformly throughout the cell. This organelle transport involves microtubule- and actin-dependent motors and is regulated by extracellular stimuli that modulate levels of intracellular cyclic adenosine 3-phosphate (cAMP). We analysed melanosome dynamics in Atlantic cod melanophores under different experimental conditions in order to increase the understanding of the regulation and relative contribution of the transport systems involved. By inhibiting dynein function via injection of inhibitory antidynein IgGs, and modulating cAMP levels using forskolin, we present cellular evidence that dynein is inactivated by increased cAMP during dispersion and that the kinesin-related motor is inactivated by low cAMP levels during aggregation. Inhibition of dynein further resulted in hyperdispersed melanosomes, which subsequently reversed movement towards a more normal dispersed state, pointing towards a peripheral feedback regulation in maintaining the evenly dispersed state. This reversal was blocked by noradrenaline. Analysis of actin-mediated melanosome movements shows that actin suppresses aggregation and dispersion, and indicates the possibility of down-regulating actin-dependent melanosome movement by noradrenaline. Data from immuno-electron microscopy indicate that myosinV is associated with fish melanosomes. Taken together, our study presents evidence that points towards a model where both microtubule- and actin-mediated melanosome transport are synchronously regulated during aggregation and dispersion, and this provides a cell physiological explanation behind the exceptionally fast rate of background adaptation in fish.

The cytoskeleton in fish melanophore melanosome positioning.

Melanophore melanosomes organelles can be regulated to move and locate correspondingly to many other different organelle types. Comparing lessons from analysis of a specific melanosome distribution can, therefore, contribute to the understanding of distribution of other organelles, and vice versa. From such data, it is now generally accepted that microtubules provide directed long-distance movement, while cell peripheral movements include microfilaments. In fish melanophores, both actin and dynein exhibit counter-forces to the kinesin-like protein in maintaining the evenly dispersed state, while actin and kinesin exhibit counter-forces to dynein in many other systems. Lessons from elevating cAMP levels indicate the presence of a peripheral feedback regulatory system involved in maintaining the evenly dispersed state. Studies from dynein inhibition suggest that the kinesin-like protein involved in fish melanosome dispersal is regulated in contrast to many other systems. One would further expect melanosome transport to be regulated also on actin/myosin, in order to prevent actin-dependent capture of melanosomes during the microtubule-dependent aggregation and dispersion. General findings will be discussed in comparison with positioning and movement of other organelle types in cells. Finally, recent data on melanosome-dependent organising of microtubules show that dynein is involved in nucleating microtubules extending from melanosome aggregates in melanophore fragments.