Ultrastructure and Function of Cephalopod Chromatophores

SYNOPSIS. Each chromatophore organ consists of a pigment celland of several radial muscle fibers that represent separatecells. The pigment granules are contained within an elasticsacculus within the pigment cell. The sacculus is attached aroundthe equator of the chromatophore to the cell membrane by zonalhaptosomes. In turn, the cell membrane is attached to the radialmuscle fibers by a dense basal lamina. The cell membrane ofthe retracted chromatophore is highly folded. Contraction ofthe radial muscle fibers is initiated by (a) excitatory junctionpotentials, (b) miniature potentials, or (c) spike potentials.The latter arise spontaneously in the muscle fibers when thesehave undergone some internal (metabolic?) change. The contractionof the muscle fibers causes expansion of the pigment-containingsacculus. Relaxation of the muscle fibers permits the sacculusto assume its original lenticular or near-spherical shape; theenergy for this is stored within theexpanded elastic componentsof the sacculus. In normal skin the chromatophore organs areentirely under the control of the central nervous system, themuscle fibers being activated only by local, excitatory postsynapticpotentials initiated by motor nerve impulses. That postsynapticpotentials are non-propagating insures that individual motorfibers can be activated individually, thus permitting a delicatecontrol of skin color by recruitment as well as by frequency.Tonic contractions and pulsations, involving spontaneous releaseof transmitter from nerve terminals and spike generation withinthe muscle fibers, respectively, are the result of altered,abnormal conditions within the skin.     

On the fine structure of the Octopus iris

Summary The Octopus iris is composed of five different layers: A, the external epithelium; B, the chromatophore layer; C, the iridocyte layer; D, the layer of muscles and collagen strands; E, the pigment epithelium. The nerves innervating the sphincter and the chromatophore muscles are identified and their neuromuscular junction is described. The motor endings of chromatophore nerves have an additional ending in presynaptic position which probably functions as a modifier of neuromuscular transmission. The chromatophores are naked and exhibit a tubular channel system between plasmalemma and pigment container which looks similar to the T-system of muscle cells.The financial support of this investigation by the Swiss National Foundation is gratefully acknowledged.     

12-(9-Anthroyl)stearic acid, a fluorescent probe for the ubiquinone region of the mitochondrial membrane.

1. 12-(9-Anthroyl)stearic acid can be incorporated into mitochondrial membranes. 2. The fluorescence properties of the membrane-bound probe are different from those of the free molecule. 3. The intensity of emission and fluorescence life-time of the probe is enhanced when, in the presence of substrate, the electron-transport chain is reduced. 4. This change in intensity has been demonstrated to be a result of collisional quenching by oxidised ubiquinone in the oxidised membrane but not when the respiratory chain is in the reduced state. 5. In pulsing anaerobic mitochondria with oxygen the rate of the fluorescence change is found to be slower than the rate of ubiquinone oxidation, suggesting that the probe detects a structural transition in the mitochondrial inner membrane. 6. This transition results in a constraint on ubiquinone motion in the reduced system. Model experiments, using lipid dispersions, have been carried out to test some of the interpretations.     

Kinetics and yields of bacteriochlorophyll fluorescence: redox and conformation changes in reaction center of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Induction of the bacteriochlorophyll fluorescence under rectangular shape of intense laser diode illumination (1 W cm(-2), 808 nm) was measured over wide time range from 10 mus to 4 s in whole cells, chromatophore and isolated reaction center protein of wild type and carotenoid-less mutant (R-26.1) of purple photosynthetic bacterium Rhodobacter sphaeroides. While the antenna-containing species showed large and positive variable fluorescence (F (v)) to initial fluorescence (F (0)) (F (v)/F (0) approximately 4.5 in whole cells), the isolated RC had negative change (F (v)/F (0) approximately -0.6) during photochemistry. In chromatophore from R-26.1, only seven times higher rate was measured than in isolated reaction center under identical experimental conditions. The enhancement effect of large antenna on the rate of photochemistry in chromatophore was partially compensated by the favorable pigment absorption properties in isolated RC. The transition from membrane bound to isolated form of the reaction center was probed by titration of zwitterionic detergent LDAO in chromatophore, and at 0.03% LDAO concentration, sharp change of the variable fluorescence was observed. The sudden drop was explained by the formation of LDAO micelles. After the photochemical phase, additional change of fluorescence yield could be observed in isolated RC considered as manifestation of long-living conformations of the trapped redox states of the protein characterized by non-exponential kinetics. Strong support was provided for use of the fluorescence induction to track structural and conformation changes at their earliest phases in chromatophores and isolated reaction centers.     

Slow reversible bleaching and fluorescence quenching caused by carotenoid absorption in chromatophores and cells of Chromatium vinosum

Light absorbed by carotenoids in Chromatium can result in photobleaching of bacteriocholorophyll and quenching of B 890 fluorescence, whereas light of the same intensity absorbed by bacteriochlorophyll has no such effect. Photobleching and fluorescence quenching are partly and slowly reversible in the dark. They are prevented by removal of oxygen or by addition of various reductants and decreased by addition of NaN₃₋ histidine or tryptophane. This suggests the participants of singlet excited oxygen in the measured phenomena. As change in temperature between 30° and –30°C and addition of gramicidin S, which changes the permeability of the membranes, does not affect photobleaching or fluorescence quenching markedly, enzymatic or structural properties do not seem to be involved. The results suggest that light absorbed by carotenoids is partly transferred to bacteriochlorophyll and partly used to excite carotenoids to triplet states. The latter process will counteract the function that carotenoids have in protecting chromatophore components against photobleaching.     

Measurement of transmembrane potentials in Rhodospirillum rubrum chromatophores with an oxacarbocyanine dye

The fluorescent dye 3,3-dipentyloxacarbocyanine (OCC) can be used as a fluorescence probe to measure transmembrane potentials across Rhodospirillum ruburm chromatophore membranes. A reversible fluorescence increase is observed in the light which is sensitive to inhibitors, permeable ions and uncouplers.Partial interchangeability between the electrical potential and the proton concentration gradient has been demonstrated by measurement of the fluorescence increase with OCC and the fluorescence quenching with 9-aminoacridine.OCC fluorescence changes can be induced also in the dark by injection of permeable salts and by rapid pH changes presumably indicating diffusion potentials. Using salt-induced diffusion potentials for calibrating the light signals and with several assumptions, the light-induced potentials were estimated as 170 mV for the maximal signal and 90–110 mV at the steady state.OCC has been shown to apparently increase the electrical conductivity of the chromatophore membrane, a fact which may be relevant to the mechanism of action of this probe.A red shift in the OCC absorption spectrum occurs when mixed with chromatophores, with a difference spectrum maximum at 495 nm. The absorption changes at 495 nm taking place in the light are similar in kinetics to the fluorescence changes. The absorbance spectrum of OCC in organic solvents is red shifted and the extent of the shift depends on the hydrophobicity of the medium. The difference spectrum compared to water in sec-butyl $\text{acetate}\text{n-}\text{hexane}$ (3 : 1, v/v) with a dipole moment of 5 was nearly identical to that of chromatophore-associated dye.The uncoupling properties of OCC at high concentrations and some difficulties in calibration limit the usefulness of this probe for quantitative measurements of transmembrane potentials.     

The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves

It has been suggested previously that non-photochemical quenching of chlorophyll fluorescence is associated with a decrease in the rate of photosystem 2 (PS 2) photochemistry. In this study analyses of fluorescence yield changes, induced by flashes in leaves exhibiting different amounts of non-photochemical quenching of fluorescence, are made to determine the effect of non-photochemical excitation energy quenching processes on the rate of PS 2 photochemistry. It is demonstrated that both the high-energy state and the more slowly relaxing components of non-photochemical quenching reduce the rate of PS 2 photochemistry. Flash dosage response curves for fluorescence yield show that non-photochemical quenching processes effectively decrease the relative effective absorption cross-section for PS 2 photochemistry. It is suggested that non-photochemical quenching processes exert an effect on the rate of PS 2 photochemistry by increasing the dissipation of excitation energy by non-radiative processes in the pigment matrices of PS 2, which consequently results in a decrease in the efficiency of delivery of excitation energy for PS 2 photochemistry.     

Different Sensitivities to Oxygen Between Two Strains of the Photosynthetic Green Sulfur Bacterium Chlorobium vibrioforme NCIB 8327 with Bacteriochlorophyll c and d

Two sub-strains of the anoxygenic photosynthetic green sulfur bacterium Chlorobium vibrioforme NCIB 8327 were derived from the same clone and could be discriminated only by their possession of either bacteriochlorophyll (BChl) c or d as the major pigment in the peripheral light-harvesting antenna system, chlorosome (Saga Y et al. (2003) Anal Sci 19: 1575–1579). In the presence of a proper amount of oxygen in the initial culture medium, the BChl d strain showed longer retardation on its growth initiation than the BChl c strain, indicating that the latter was advantageous for survival under aerobic light conditions which produced reactive oxygen species in vivo. The result would be ascribable to the difference of the midpoint potentials between two kinds of chlorosomes formed by self-aggregates of BChl c and d as measured by their fluorescence quenching.     

Characterisation of the effects of Antimycin A upon high energy state quenching of chlorophyll fluorescence (qE) in spinach and pea chloroplasts

High energy state quenching of chlorophyll fluorescence (qE) is inhibited by low concentrations of the inhibitor antimycin A in intact and osmotically shocked chloroplasts isolated from spinach and pea plants. This inhibition is independent of any effect upon pH (as measured by 9-aminoacridine fluorescence quenching). A dual control of qE formation, by pH and the redox state of an unidentified chloroplast component, is implied. Results are discussed in terms of a role for qE in the dissipation of excess excitation energy within photosystem II.Abbreviations 9-AA_max = Maximum yield of 9-aminoacridine fluorescence - DCMU = 3(3,4-dichlorophenyl)-1,1-dimethylurea; F_max ± Maximum yield of chlorophyll fluorescence - hr = hour - PAR = Photosynthetically Active Radiation - Q_A = Primary stable electron acceptor within photosystem II - qE = High energy state quenching of chlorophyll fluorescence - qI = quenching of chlorophyll fluorescence related to photoinhibition - qP = Quenching of chlorophyll fluorescence by oxidised plastoquinone - qQ = photochemical quenching of chlorophyll fluorescence - qR = (F_max—maximum level of chlorophyll fluorescence induced by the addition of saturating DCMU) - qT = Quenching of chlorophyll fluorescence attributable to state transitions     

Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q(A)) of photosystem (PS) II. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 micros to 5 s). The about 20% lowering of the maximum fluorescence yield F(M), observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH(2) by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH(2) starts getting reoxidized by PS I activity. NAD(P)H-dependent restoration of F(M) was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl(2) that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F(M). Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F(0) allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F(0) level (Q(0)) and to compare it with the fractional quenching at the F(M) level (Q(M)). The experimentally determined Q(0)/Q(M) ratios were found to be equal to the corresponding F(0)/F(M) ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve. Changes in the redox state of Photosystem II electron acceptors and fluorescence emission from Photosystems I and II

An analysis of the photo-induced decline in the in vivo chlorophyll a fluorescence emission (Kautsky phenomenon) from the bean leaf is presented. The redox state of PS II electron acceptors and the fluorescence emission from PS I and PS II were monitored during quenching of fluorescence from the maximum level at P to the steady state level at T. Simultaneous measurement of the kinetics of fluorescence emission associated with PS I and PS II indicated that the ratio of PS I/PS II emission changed in an antiparallel fashion to PS II emission throughout the induction curve. Estimation of the redox state of PS II electron acceptors at given points during P to T quenching was made by exposing the leaf to additional excitation irradiation and determining the amount of variable PS II fluorescence generated. An inverse relationship was found between the proportion of PS II electron acceptors in the oxidised state and PS II fluorescence emission. The interrelationships between the redox state of PS II electron acceptors and fluorescence emission from PS I and PS II remained similar when the shape of the induction curve from P to T was modified by increasing the excitation photon flux density. The contributions of photochemical and non-photochemical quenching to the in vivo fluorescence decline from P to T are discussed.     

Skin color in the squids Loligo pealii and Loligo opalescens

Summary Rapid, physiological color changes seen in the skin of cephalopods are due to a unique anatomical system composed of chromatophore organs and iridophores. The morphology and ultrastructure of the chromatophores was studied in the squids Loligo pealii Lesueur and Loligo opalescens Berry. A three-dimensional model of a brown chromatophore was reconstructed from serial sections for the electron microscope.The chromatophore organ is composed of a central nucleated pigment cell, 10–30 obliquely striated muscle cells (radially arranged on the equator of the pigment cell), axons, Schwann cells, and sheath cells. The pigment cell consists of a central aggregation of pigment granules and surrounding peripheral cytoplasmic compartments. These regions are incompletely separated by an electron-dense, sac-like structure, the pigment container. Proximal portions of a muscle cell contact the pigment cell in regions called myo-chromatophore junctions. Neuromuscular and myo-muscular junctions are also present.The results presented are discussed in terms of previous morphological and physiological studies of chromatophores.Part of a study submitted in partial fulfillment of the requirement for the degree of Ph. D. (Anatomy), the Graduate School of Basic Medical Sciences, New York Medical College, New York, N.Y. 10029.The research reported here was in part supported by grants from the Health Research Council of the City of New York (U-1008) and United States Public Health Service, General Research Grant No. FR-05398.Report on some of this material was given at the Annual Meeting of the American Association of Anatomists, Philadelphia, Pennsylvania, April 19–22, 1971.     

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for ¹O₂ quenching by BChl molecules (k_q ^M) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the k_q ^M values coincide with the real values for ¹O₂ quenching rate constants by BChl molecules. Aggregation weakly influenced the k_q ^M values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the k_q ^M value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus ¹O₂ quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of ¹O₂ formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as ¹O₂ physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.Abbreviations BChl bacteriochlorophyll - MBpd methyl bacteriopheophorbide - Chl chlorophyll - TPP meso-tetraphenylporphyrin - TPPS meso-tetra (p-sulfophenyl) porphyrin     

The morphology of dermal chromatophores in the infrared-reflecting glass-frog Centrolenella fleischmanni

The morphology and organization of chromatophores in the neotropical glass-frog, Centrolenella fleischmanni (family Centrolenidae), were studied with both light and electron microscopes. Four types of pigment cells are described in the dorsal skin. The fine structure of two chromatophores corresponds to the typical amphibian xanthophore and iridophore; one is similar to the unusual melanophore found in phyllomedusine hylids; the fourth cell type is unlike any chromatophore previously described. Pigment granules in the unusual chromatophore are moderately electron-dense and have an irregular shape, suggesting a fluid composition. This pigment appears to be laid down in organelles similar in appearance to pterinosomes. The organization of pigment cells in this species differs from that of other green, leaf-sitting frogs in that there are few discrete groups resembling “dermal chromatophore units.” It is suggested that the unusual new pigment cell contributes significantly to the overall green color of C. fleischmanni.     

The use of zinc(II) to probe iron binding and oxidation by the ferritin (EcFtnA) of Escherichia coli

 The ferritin of Escherichia coli (EcFtnA) is similar to human H-chain ferritin (HuHF) in having 24 subunits, each containing a dinuclear site at which two iron atoms can be oxidised (the diiron centre). In EcFtnA, unlike HuHF, fluorescence quenching of Trp122, located near site A of the dinuclear centre, can be used to monitor metal binding (this tryptophan is absent from HuHF). Metal binding also perturbs the UV absorbance spectrum of Trp122 and that of Tyr24 (a conserved residue near site B of the dinuclear centre). Using UV-difference spectroscopy and fluorescence quenching it is shown that Fe(II) and Zn(II) bind at the same sites, A and B. Sequential stopped-flow studies of Fe(II) binding and oxidation also show that Zn(II) is an effective competitor of Fe(II) binding and an inhibitor of its oxidation. Received: 10 June 1998 / Accepted: 18 September 1998     

Spectral Studies of a Chlorophyll Pigment with Fluorescence Maximum at 698 mμ

A chlorophyll type pigment (F698) fluorescing maximally at 698 mμ at 77°K has been observed in preparations of chlorophyll. This fluorescence is quenched by small amounts of naturally occurring materials, including plastoquinone and the ubiquinones, and by nitrobenzene, probably by formation of a nonfluorescent complex. Fluorescence quenching does not occur in the presence of carotenes, xanthophylls, or reduced plastoquinone and ubiquinone. The fluorescence is sharply temperature dependent, with a steep rise in intensity occurring at 165°K. At 77°K the fluorescence yield is between 0.8 and 1.0. The red absorption maximum of the pigment is at 675 mμ at room temperature and at 688 mμ at 77°K. In vivo, a low temperature emission is also observed at 698 mμ, and this fluorescence is quenched by nitrobenzene. It is proposed that the pigment found in vitro is also the one responsible for emission at 698 mμ in vivo. A reaction of F698 with plastoquinone is suggested as the primary photochemical step in system II of photosynthesis.     

Chromatophore radial muscle fibers anchor in flexible squid skin

Cephalopod skin is soft, flexible, and produces rapid color changes for camouflage and signaling primarily by regulating the shapes of its numerous chromatophore organs. Each chromatophore has 10–30 radial muscle cells, termed fibers, under central nervous system control. Each fiber contains myofilaments that contract in concert to stretch the pigment‐containing cell from its punctate, spherical state to a fully expanded thin disk of color. Expansion occurs in less than one second and can result in a 14‐fold expansion in pigment cell diameter. We investigated the anchoring mechanism of radial muscle fibers that expand pigment cells in the longfin squid, Doryteuthis (Loligo) pealeii. The proximal Active Zone of a radial muscle fiber adheres to the pigment cell within an ensheathing sinus. The distal portion forms terminal arbors, thereby increasing the surface area, to adhere it to the dermal extracellular matrix (ECM). While the muscle fiber is attached to the pigment cell with haptosomes, the remainder of the fiber is adhered to the surrounding basal lamina (part of the ECM) by numerous, closely spaced, small costamere‐like projections. Branching of the radial muscle fiber termini and the costamere‐like attachments are key anatomical specializations that anchor the radial muscle fibers in the pliable skin while allowing the freedom of movement required for large changes in pigment cell diameter. We postulate that these features may be relevant for the development of soft actuation models in materials science.     

Cellular Aspects of the Control of Physiological Color Changes in Crustaceans

SYNOPSIS. Red chromatophores(erythrophores) of the prawn, Palaemonetesvulgaris, are controlled by pigment—dispersing and -concentratinghormones. Recent experiments on the modes of action of thesehormones are described, followed by a theory which satisfactorilyexplains the data. Red pigment-concentrating hormone is dependentupon sodium ions for a strong response to occur. There is asimilar dependency of red pigment—dispersing hormone uponcalcium ions. Ouabain inhibits the response to red pigment—concentratinghormone; tetrodotoxin enhances it. Erythrophores with maximallydispersed pigment had a transmembrane potential of 55±15mv inside negative in one series of experiments and 56±4mv in another. No appreciable changes in permeability occurwhen depolarizing and hyperpolarizing currents are passed througha microelectrode within the chromatophore. Red pigmentconcentratinghormone causes hyperpolarization of the transmembrane potential.The magnitude of hyperpolarization is directly related to thedegree of pigment concentration. Adenosine 3`;, 5`-monophosphate(cyclic AMP) causes dispersion of the red pigment but has nopigment-concentrating effect. The primary action of red pigmentconcentratinghormone is most likely stimulation of a pump which exchangessodium ions from inside the chromatophore with potassium ionsfrom the outside, whereas red pigment-dispersing hormone quitelikely stimulates entry of calcium ions into the chromatophore.     

Effects of exogenous guanosine on chromatophore differentiation in the axolotl

Guanosine is shown to dramatically alter the pigment phenotype of axolotls by suppressing melanization and enhancing the biosynthesis and deposition of purine-derived pigments. Phenotypic changes caused by guanosine are manifested by altered chromatophore differentiation patterns such that few black pigment cells (melanophores) differentiate (and those that do are punctate and necrotic in appearance), whereas the development of yellow (xanthophore) and reflecting (iridophore) pigment cells is enhanced. Mechanisms for changes in chromatophore differentiation, and thus pattern formation, are discussed, including the possibility that pigment cells may undergo transdifferentiation in vivo.