Pigment Movement Research Articles

The blockade of pigment displacement in swordtail erythrophores by microinjection of antiactin antibody

AbstractWhen antiactin antibody produced against carp skeletal muscle actins was microinjected into monolayer‐cultured swordtail erythrophores, pigment displacement was almost completely blocked. Moreover, the injected antibody was equally effective at blocking the aggregation of dispersed pigment and the dispersion of aggregated pigment. If antibody saturated with carp actins was microinjected, no such blockade occurred.Microinjection of phalloidin or DNase I into these cells brought about an incomplete inhibition of their pigment displacement. These two drugs tended to inhibit pigment dispersion more than aggregation, suggesting that actins are differently involved in the two phases of pigment movement. The results all indicate that actins are functional elements in the pigment displacement of swordtail erythrophores, and that G‐F transformations of these molecules play little or no role in pigment aggregation, but are implicated in some way in dispersion.

Regulation of Release and Mode of Action of Crustacean Chromatophorotropins

Pigment movements in crustacean chromatophores are regulated by pigment-concentrating and pigment-dispersing neurosecretory hormones. The release of these hormones from the neurosecretory system is thought to be controlled by neural signals. Although the exact cellular sources of chromatophorotropins and the nature of synaptic input to the neurosecretory system remain unresolved, recent pharmacological studies on fiddler crabs indicate that certain biogenic amines contribute to the release of some of the chromatophorotropins. The current evidence suggests that: 5-hydroxytryptamine stimulates the release of RPDH (red pigment dispersing hormone), dopamine triggers the release of RPCH (red pigment concentrating hormone), and norepinephrine stimulates the release of BPDH/MDH (black pigment/melanin dispersing hormone). Nothing is known of the regulation of release of leucophorotropins and xanthophorotropins. Like most other peptide hormones, crustacean chromatophorotropins are thought to exert their effect by first binding to receptors on the cell surface and then triggering a series of events leading to cellular responses, but the details are far from clear. Recent work suggests that hormone-induced pigment movements may be modulated/mediated by alterations in cyclic nucleotide levels and/or by changes in Ca2+ distribution or flux. Both cyclic nucleotides and Ca2+ may modulate motility by regulating the integrity and/or function of cytoskeletal elements, but the details of this mechanism and the precise roles of cytoskeletal elements in the pigment-translocating process remain unresolved.

Cellular Aspects of Hormonally Controlled Pigment Translocations Within Chromatophores of Poikilothermic Vertebrates

Pigment movements triggered by the vertebrate melanocyte-stimulating hormone (MSH) are known to be mediated at the cellular level by cyclic adenosine monophosphate (cAMP). A similar role for cAMP is indicated in the melanosome-dispersing action of catecholamines acting via rβ3-adrenergic receptors. Thus, MSH and rβ3-adrenergic agonists cause an elevation of cAMP within chromatophores. On the other hand, a lowering of cAMP level seems to be associated with the paling action of melatonin and α-adrenergic agonists. Depending upon the cell type and species tested, cyclic guanosme monophosphate (cGMP) may have no effect or cause either darkening or paling. Although cGMP induces paling in frog skin, the lack of evidence for a physiological paling agent makes it difficult to assign a role for cGMP The mechanism of action of cyclic nucleotides and the details of the pigment-translocating mechanism are far from clear, but recent studies indicate that changes in free calcium ion levels play a critical role. This article will primarily review three areas' the paling action of cGMP, the darkening action of ionophore A23187, and the actions of enkephalms and endorphins on chromatophores. In addition, new data are presented to support the concept that the A23187-induced darkening of frog skin does not involve either prostaglandin biosynthesis or adenylate cyclase stimulation.

The fine structure of the compound eye of the African armyworm moth, Spodoptera exempta Walk. (Lepidoptera, Noctuidae).

The morphology of the compound eye of the noctuid moth Spodoptera exempta was investigated by electron microscopy. This eucone superposition eye is composed of about 8000 ommatidia. Each ommatidium is surrounded by six secondary pigment cells showing pigment movement according to the state of adaptation. It contains four crystalline cone cells forming together a crystalline cone and tract, two primary pigment cells, which encompass the crystalline cone, and usually eight retinula cells. On the basis of their rhabdomeric structure, three types of retinula cells can be distinguished. According to the structure of the rhabdom, two types of ommatidia are found in different regions of the eye. The rhabdom of the lobed type, providing more than 80% of ommatidia, is composed of V-shaped rhabdomeres with fanwise arranged microvilli. The rhabdom of the square type, found in a small area in the dorsal region of the eye, consists of triangular rhabdomeres with parallel microvilli. The functional significance of this difference is discussed.

Diurnal changes in angular sensitivity of crab photoreceptors

The electrophysiological and anatomical consequences of diurnal changes in screening pigment position were investigated in the apposition eye of the portunid crabScylla serrata. Intracellular recordings revealed that the acceptance angles of dark-adapted photoreceptors enlarged up to four-fold at night compared with photoreceptors dark-adapted in the day. Furthermore, while light adaptation at night caused acceptance angles to narrow, dark adaptation in the day caused no significant broadening of angles. These electrophysiological changes correlated with pigment movements in the eye observed both histologically and in the deep pseudopupil. It is found that the distal pigment cells change diurnally so that the field-stop which these cells form in front of the photoreceptors is opened in the night and closed in the day time. One feature of the diurnal rhythm is that it prevents photoreceptor fields of view enlarging when eyes are dark adapted in the day. InScylla, photoreceptor fields of view take tens of minutes to narrow upon exposure of crabs to light at night. By preventing a similar broadening in the day, the diurnal rhythm may enable animals suddenly leaving dark refuges to be pre-adapted to daylight. To a range of species which utilise refuges such a mechanism would be of significant advantage, especially after disturbance by predators.

Light induced retinal screening pigment migration independent of visual cell activity

1. The migration of the granules in the retinal screening pigment cells was studied in the compound eye of the mothDeilephila elpenor. Pigment position was determined by observing the size of the ‘glow’ caused by reflection of incident light. 2. The spectral efficiency of the pigment migration was tested by wavelengths between 352 and 608 nm. Maximal pigment movement was seen after exposure to wavelengths between 352 and 443 nm. The movement decreased with wavelength between 443 and 530 nm. (Maximal spectral sensitivity, and efficiency, of the visual cell layer is to 525 nm). 3. Exposure to white light caused pigment expansioninspiteofclampingthe visual cell receptor potential. 4. Blue (λ = 458 nm) light elicited pigment expansion also after the screening pigment cell layer had been isolated from the visual cells. 5. In animals dying in darkness the screening pigment remained in the energy consuming contracted position while the body had already become completely stiff. The results suggest that: a) photosensitive system, maximally sensitive to UV and blue wavelengths, is located within the screening pigment cells, and that absorption of photons by this system directly interferes with the energy consuming mechanism which keeps the pigment contracted. Alternatively, pigment expansion is triggered by the UV (λmax 350 nm) and violet (λmax = 450 nm), but not by the green (λmax = 525 nm), sensitive visual cells. 7. moths are trapped by UV wavelengths because these wavelengths elicit pigment expansion. Dim ambient light is then absorbed by the screening pigment, and the animal flies towards the trap which is the only light visible to the animal. During sunset the pigment migrates to the dark-adapted position as soon as the intensity of the short wavelength light is sufficiently low, whereby discrimination of variations in ambient light intensity is enhanced.

Hormone-induced filopodium formation and movement of pigment, carotenoid droplets, into newly formed filopodia.

Treatment of cultured goldfish xanthophores by hormone (ACTH) or c-AMP induces not only pigment dispersion, but subsequent outgrowth of processes, and pigment translocation into these processes. These latter effects are shown to proceed as follows: First the edge of the cytoplasmic lamellae takes on a scalloped contour with numerous protrusions. These presumably serve as nucleation centers where short microfilament bundles are assembled. Later, the microfilament bundles elongate ("grow"), often resulting in an extension of the protrusions to become filopodia while the proximal end of the microfilaments penetrates into the thicker portion of the cellular process which now houses the pigment, i.e., the carotenoid droplets. Carotenoid droplets appear to migrate along the microfilament bundles, or cytoplasmic channels associated with them, into the filopodia. Finally, some of the filopodia become broader, thicker and laden with carotenoid droplets and are then recognized by light microscopy as pigmented cellular processes. The microfilaments have been shown to be actin filaments by their thickness, the size of their subunits, and decoration by heavy meromyosin. Evidence is presented which suggests that the growth of these actin filaments may come about by recruitment from short F-actin strands found in random orientation in adjacent areas.

The control of pigment migration in isolated erythrophores of Holocentrus ascensionis (Osbeck). I. Energy requirements

Erythrophores isolated from the scales of the marine teleost, Holocentrus ascensionis (Osbeck), are capable of rapidly aggregating or dispersing numerous red pigment granules within their cytoplasm by translocating them along radial paths delineated by bundles of radially oriented microtubules. Pigment translocation is accompanied by transformations in the morphology of the cytoplasmic matrix, or microtrabecular lattice (MTL), in which the pigment granules are suspended. It appears that the MTL as a whole contracts toward the cell center during aggregation, carrying the pigment granules inward along with it, and is restructured during dispersion, using the radial microtubules as guides. We examined the energy requirements of pigment migration and the accompanying MTL transformations. Cellular ATP was depleted using the specific metabolic inhibitors 2,4 dinitrophenol, NaCN and oligomycin. All three of these drugs, which inhibit oxidative phosphorylation by different mechanisms, prevent both pigment dispersion and MTL transformation to dispersed morphology, while aggregation is unaffected. Inhibitor-treated cells recover normal pigment movements and MTL morphology when inhibitor is washed out of the cells with fresh medium. Potential energy apparently is stored in the MTL by some ATP-dependent process during dispersion and is converted to kinetic energy during aggregation. The results of this study strengthen the hypothesis that the MTL, working in concert with the radial microtubules, is the vehicle for pigment translocation in the erythrophore system.

Spectral sensitivity and retinal pigment movement in the crab Leptograpsus variegatus (fabricius).

1. The retina of Leptograpsus contains five types of movable screening pigment. The positions of these were found under various conditions of illumination in the day and at night. 2. Intracellular recordings were made of the spectral responses of retinula cells R1-7 under the same conditions, with the eye in situ. 3. The spectral absorptions of the individual screening pigments were measured by ultramicrospectrophotometry. 4. Calculations based on a simple model of screening pigment action suggest that the observed variation in spectral sensitivity with light and dark adaptaton may be largely explicable in terms of the effects of these screening pigments on a rhodopsin of peak absorbance at 485 nm. 5. Light-adapted angular sensitivities are comparable to those of insects with high acuity apposition eyes.

Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements.

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

Insect pupil mechanisms

The light-dependent pigment migration system of dragonfly ocelli was studied by optical, non-invasive techniques. The median ocellus is comprised of two lateral halves, as can be demonstrated in the intact animal since illumination of the receptors in one half of the median ocellus only induces a movement of pigment located in that half. Measurable pigment migration can occur within a few seconds, but its speed and extent depends on light intensity. Dispersal of pigment, which occurs upon light adaptation, proceeds faster than retraction, which occurs upon dark adaptation. Action spectra for pigment movement have been determined in Sympetrum and Anax. The spectrum for Sympetrum has a prominent UV peak, moderate blue sensitivity, and very low green sensitivity. A similar profile is obtained in Anax, but only after intense orange adaptation which suppresses the green sensitivity. The results conform to the known spectral sensitivities of Libellulid and Aeschnid ocellar receptors. It is concluded that the photoreceptors drive pigment movement through an unknown mechanism. The effect of the migration of pigment is the selective reduction of radiant flux on the retina from luminous sources at high elevations relative to the animal's normal flying posture.

Stereo high voltage electron microscopy of melanophores: Matrix transformations during pigment movements and the effects of cold and colchicine

Pigment migration in isolated melanophores of the angelfish, Pterophyllum scalare, has been studied by high voltage electron microscopy. Cells were isolated from the scales by collagenase and allowed to spread on Formvar and carbon-coated gold grids. Melanophores were then fixed by glutaraldehyde and osmium tetroxide and critical-point dried for viewing of whole cells in a high voltage electron microscope (1000 kV). The three-dimensional organization of the cytoplasmic matrix was stereoscopically examined in different states of pigment distribution, as well as under cold and colchicine treatment. The most prominent matrix constituent is an extensive mesh of cytoplasmic filaments (microtrabeculae) 2–18 nm in diameter that make contact to microtubules, pigment granules, and mitochondria. Microtrabeculae undergo dramatic changes in structural appearance in association with different phases of pigment movements. Cells fixed in the process of pigment aggregation are characterized by thickened and beaded trabeculae which may form irregular clots. Part of this material trails behind centripetally moving melanosomes. In dispersing cells, microtrabeculae are straight and of relatively uniform thickness throughout their length and form a highly ordered three-dimensional lattice. Reconstruction of the mesh in part precedes the arrival of pigment granules. Under the influence of cold or colchicine treatment, microtrabeculae show a high degree of polymorphism, being beaded, branched, or flattened with globose ends. Rather formless heaps are found associated with the surface of pigment granules. Since, however, these treatments also remove microtubules, the other important component of the cytoplasmic frame, alterations in microtrabecular structure may simply be mediated through removal of this organelle. In an attempt to separate the effects on microtrabeculae and microtubules from one another, cells have been cold-treated for only 15 min, a procedure that leaves a considerable portion of microtubules intact. Also under these conditions, microtrabeculae are beaded or transformed to globose heaps and flattened sheets. The observations suggest an involvement of microtrabeculae in the process of granule movement. Centripetal melanosome migration thereby seems associated with a collapse of microtrabeculae which again are reconstructed during pigment dispersion. The cold and colchicine experiments indicate direct effects of these agents on the structure and possibly also the function of the trabecular mesh. The significance and possible chemical composition of microtrabeculae is discussed.

Melanophore potentials of the chromatically intact spinal stoneloach (Noemacheilus barbatulus L.) following adaptation to varying backgrounds

1. The transient colour response of the stoneloach (Noemacheilus barbatulus) is similar to that described for some other freshwater teleosts. 2. A method is described for measuring the melanophore transmembrane potentials resulting from direct activity of the intact chromatic system. 3. Using this method the melanophore index and the melanophore potential at the cell centrosphere were measured in fish adapted to black and white backgrounds and during background reversal (Figs. 5, 6). 4. The amount of pigment movement for the same change in centrosphere potential is greater in melanophores that are aggregating than in those that are dispersing. 5. The melanophore index and the melanophore potential at the cell centrosphere were measured in fish subjected to darkness following adaptation to black and white backgrounds (Fig. 7), and in fish subjected to illumination on black and white backgrounds after adaptation in darkness (Fig. 8). 6. The melanophore index and the melanophore potential both at the cell centrosphere and at the edge of the pigment were measured in fish adapted to backgrounds of varying reflectivity (Table 1). In all positions of the pigment the sum of these potentials remained nearly constant, although the potentials themselves varied (Figs. 9, 10).

COLCHICINE AND CYTOCHALASIN B: A FURTHER CHARACTERIZATION OF THEIR ACTIONS ON CRUSTACEAN CHROMATOPHORES USING THE IONOPHORE A23187 AND THIOL REAGENTS

1. Using the prawn, Palaemonetes pugio, and the fiddler crab, Uca pugilator, the modes of action of cytochalasin B and colchicine in inhibiting pigment granule movement were further characterized.2. Colchicine was unable to inhibit, in ovarian erythrophores of Palaemonetes pugio, pigment concentration caused by the ionophore A23187. It did, however, inhibit somewhat the pigment dispersion that normally occurs upon placing A23187-treated erythrophores in Ca++-free, EGTA-containing saline.3. Inhibition by colchicine of pigment concentration in melanophores of Uca pugilator was not prevented by high concentrations of glutathione or cysteine hydrochloride. Pigment concentration was inhibited by mersalyl acid, and this was prevented by glutathione.4. Cytochalasin B inhibited ionophore A23187-induced pigment concentration in ovarian erythrophores of Palaemonetes pugio.5. Electron microscopy revealed a filamentous matrix interconnecting the pigment granules of Uca pugilator.6. Speculation is presented as to the modes of action of cytochalasin B and colchicine, and a heuristic model for the control of pigment movement is put forward.

A microtubule-independent component may be involved in granule transport in pigment cells

COLOUR change in lower vertebrates is based on the movement of thousands of pigment granules within integu-mental chromatophores. In some types of pigment cells, particularly those of teleost fish, pigment aggregation in the cell centre and redispersion into the cell processes may take place within fractions of a minute1,2. These cells may therefore be a suitable model system for the study of intra-cellular vesicle transport. There is considerable information about the structure of chromatophores, in particular their prominent microtubule system2–5, but the mechanism of pigment movements remains obscure. Pigment granule transport is thought to be microtubule-dependent6 and microtubules definitely play a part in this process1,2,6. However, it remains to be determined whether these organelles also directly provide the driving force for the movements. The crucial question is: what happens when the microtubule frame is withdrawn experimentally? We therefore tested the ability of chromatophores to move pigment granules in conditions where microtubules were absent, and further investigated the nature of the physical relationship between microtubules and pigment granules. We now report the presence of a second component involved in the transport process.

The optic nerve mediates the circadian pigment migration in the median eyes of the scorpion

1. 1. The peripheral visual pathway from the median eyes of the scorpion Androctonus australis was interrupted at different points and the effect on the circadian rhythm of median-eye sensitivity was examined. 2. 2. Any interruption of the visual pathway distal to the supraesophageal ganglion abolishes the circadian sensitivity rhythm in the median eyes. This rhythm is thus controlled by efferents in the optic nerve (very probably via the neurosecretory axons) rather than by way of the hemolymph. 3. 3. Following transection of the optic nerve, the sensitivity of the median eyes proceeds rapidly to the daytime state. This condition is associated with movement of the screening pigment into the distal ends of the visual cells. 4. 4. The oscillator system controlling the circadian pigment migration in the median eye cannot be located in the eye itself, but must lie in the CNS, proximal to the first optic ganglion. The oscillator itself need not be connected to both median eyes in order to function normally, as revealed by the continued rhythm in the contralateral eye following unilateral optic nerve section.

Mechanisms controlling pigment movements within swordtail ( Xiphophoprus helleri) erythrophores in primary cell culture

1. 1. Pigment organelle (pterinosomes and carotenoid vesicles) movements within isolated swordtail ( Xiphophorus helleri) erythrophores cultured in vitro were studied. 2. 2. Pterinosome and carotenoid vesicles migrate centripetally to a juxtanuclear position or centrifugally into the dendritic processes of erythrophores in response to catecholamines. 3. 3. Cateeholamine stimulation of alpha adrenoceptors results in centripetal pigment organelle migration whereas beta adrenoceptors mediate centrifugal movements. 4. 4. Melatonin also induces perinuclear aggregation of pigment organelles but by receptor mechanisms separate from adrenoceptors. 5. 5. MSH and ACTH stimulate pigment organelle dispersion. Centrifugal dispersion of erythrophore pigments is also stimulated by cyclic AMP and dibutyryl cyclic AMP.

Structure of the Eyes of the Pea Aphid, Acyrthosiphon pisum1

The 3 separate types of visual centers, the ocelli and the primary and secondary compounds eyes of the pea aphid, are illustrated and discussed. Each system is morphologically distinct and each, within the limits imposed by its own structure, should be capable of receiving visual signals and transmitting them to the brain. Differences in the location of pigments within the ommatidia of secondary compound eye of apterous viviparous females are illustrated. The exact functions of each type of eye and reasons for differences in the movement of pigment are unknown.

Light-induced alterations in cell shape and pigment displacement in chromatophores of the sea urchin Centrostephanus longispinus.

Alteration in cell shape of the ligh-sensitive chromatophores of Centrostephanus longispinus are described. Upon illumination a centrifugal pigment movement starts within extremely thin filopodia which radiate from the cell body. With continued pigment migration the cellular processes increase in length and diameter and give the cell an irregular stellate appearance. Pigment movement within the cellular processes is discontinuous in space and time and may occur independently in single filopodia. The motion of single granules shows characteristic features of a saltatory movement.

Neursecretory fibres in the median eyes of the scorpion, Androctonus australis L.

The retina of the median eyes of the North African scorpion, Androctonus australis L., is supplied with numerous neurosecretory nerve fibres which establish synaptoid contacts on the retinula cells. The number of fibres or profiles of varicosities of fibre terminals associated with a retinular unit (five retinula cells with a fused rhabdom) varies between 10 and 20. Electron-opaque vesicles with a diameter of 80-100 nm are abundant within the axonal profiles. The synaptoid junctions are characterized by postsynaptic electron-dense material on the inner leaflet of the retinula cell membrane and, frequently, presynaptic submembranous dense material. Because of these ultrastructural features, the junctions observed here resemble typical interneuronal synaptic contacts. Hence this kind of neurosecretory junction appears to be unique among arthropods. It is suggested that the neurosecretory fibres within the retina represent the efferent pathways for the control of the circadian pigment movements within the retinula cells.