Immunogold labeling of organelles in the bioluminescent dinoflagellate Gonyaulax polyedra with anti-luciferase antibody

A polyclonal antibody directed against the luciferase of the luminous dinoflagellate Gonyaulax polyedra labels both dense vesicles and trichocyst sheaths, as visualized in the electron microscope after treatment of antibody-reacted sections with an immunogold probe. Because of their similar size, shape and localization, the dense vesicles seen with the electron microscope are postulated to correspond to autofluorescent particles seen with the fluorescent microscope, which are known to be the origin of bioluminescent flashes in this alga. The explanation for the trichocyst sheath-specific labeling is less evident. The possibility that a second antibody of different specificity is involved has not been excluded but seems unlikely. Alternatively, it could be due to a different but antigenically cross-reacting protein. But the possibility that luciferase itself occurs in two different organelles is intriguing and consistent with previous biochemical studies of cell extracts.     

Role of a luciferin-binding protein in the circadian bioluminescent reaction of Gonyaulax polyedra

A luciferin-binding protein (LBP), which binds and protects from autoxidation the substrate of the circadian bioluminescent reaction of Gonyaulax polyedra, has been purified to near homogeneity. The purified protein is a dimer with two identical 72-kDa subunits, and an isoelectric point of 6.7. LBP is a major component of the cells, comprising about 1% of the total protein during the night phase, but drops to only about 0.1% during the day. The luciferin is protected from autoxidation by binding to LBP, and one luciferin is bound per dimer at alkaline pH (Ka approximately 5 x 10(7) M-1). The protein undergoes a conformational change with release of luciferin at pH values below 7, concurrent with an activation of Gonyaulax luciferase. LBP thus has a dual role in the circadian bioluminescent system.     

Purification and characterization of peridinin-chlorophyll a-proteins from the marine dinoflagellates Glenodinium sp. and Gonyaulax polyedra

Summary A peridinin-chlorophyll a-protein complex (PCP) was obtained in large quantity from the marine dinoflagellates, Glenodinium sp. and Gonyaulax polyedra. The chromoproteins have similar molecular weights, 35,500 for Glenodinium sp. and 34,500 for G. polyedra. The proteins from the PCP complex of Glenodinium sp. dissociated from the chromophore on treatment with 1% sodium dodecyl sulfate (SDS) at room temperature. The protein component was a single subunit with a molecular weight of 15,500. Proteins from the PCP complex of G. polyedra were composed of a single polypeptide with a molecular weight of about 32,000. Two peridinin-chlorophyll a-proteins from Glenodinium sp. accounted for 70% of the PCP complex and had isoelectric points of 7.4 and 7.3. The PCP complex from G. polyedra was dominated by a single chromoprotein with an isoelectric point of 7.2 Chromophore analysis indicated the presence of only peridinin and chlorophyll a in a molar ratio approaching 4:1. Other pigments characteristically found in dinoflagellates were absent. Fluorescence excitation spectra of purified PCP indicated an efficient energy transfer from peridinin to chlorophyll a, an observation that lends support to the reported role of peridinin as an accessory pigment in photosynthetic oxygen evolution. In several other brown colored dinoflagellates examined, PCP representtd less than 20% of the total peridinin. However, no PCP could be isolated from cultures of Amphidinium carterae (PY-1). This study provides further evidence that PCP is a normal component of most peridinin-containing dinoflagellates, and functions as a light-harvesting component of the dinoflagellate chloroplast. No fucoxanthin-containing analog of PCP was detected in the chrysophyte, Cricosphera carterae and the dinoflagellate Glenodinium foliaceum.Abbreviations PCP peridinin-chlorophyll a-protein complex - PCP's peridinin-chlorophyll a-proteins - SDS sodium dodecyl sulfate - pl isoelectric point - DEAE diethylaminoethyl cellulose - TLC thin layer chromatography - A optical absorbance at a designated wavelength - SIO (F.T. Haxo), Scripps Institution of Oceanography collection     

Colocalization of luciferin binding protein and luciferase to the scintillons ofGonyaulax polyedra revealed by double immunolabeling after fast-freeze fixation

Summary Two proteins,Gonyaulax luciferase and the luciferin binding protein, are involved in the bioluminescent reaction of the unicellular marine algaGonyaulax polyedra. Using antibodies raised separately against the purified proteins, their ultrastructural localizations were visualized by double immunogold labeling on sections after fast-freeze fixation, freeze-substitution and embedding in Epon or in LR White. Gold particles of two sizes attached to the secondary antibodies allowed the two primary antibodies to be distinguished. The two colocalized to cytoplasmic densifications (scintillons), which occurred in close association with the vacuolar membrane near the periphery of the cell. They also occurred in the cytoplasm of the Golgi area, either over densifications without associated membranes (prescintillons), or as very small colocalizations not associated with any evident cytoplasmic differentiation. No other site of colocalization was observed, thus unambiguously establishing the ultrastructural identity of the bioluminescent organelles.Abbreviations FFF fast-freeze fixation - FS freeze-substitution - IGS immunogold staining - LBP luciferin binding protein - PBS phosphate buffered saline - TBS tris-buffered saline Dedicated to the memory of Professor Beatrice Marcy Sweeney     

An optional dyadic junctional complex revealed by fast-freeze fixation in the bioluminescent system of the scale worm

In the bioluminescent system of the scale worm, the facilitation of the successive flashes is correlated with the progressive recruitment, in each photogenic cell, of new units of activity, the photosomes. To characterize morphologically the coupled state of the photosomes, known to decouple within seconds at rest, fast-freeze fixation was applied to stimulated and nonstimulated elytra and followed by substitution with OsO4 in acetone. The results showed striking differences. Photosomes were surrounded by a new type of smooth endoplasmic reticulum (ER) called intermediate endoplasmic reticulum (IER). In nonstimulated elytra, the IER was most often unattached in the cytoplasm. After stimulation, the IER was connected to large terminal saccules that formed dyad junctions with the plasma membrane. Most of these junctional complexes were symmetrical (triads) and occurred in front of narrow extracellular spaces. These spaces were either constitutive, like invaginations or clefts along adjacent cells and adjacent pouches, or resulted from the pairing of long pseudopods which expanded into a wide extracellular compartment and twisted together in a dynamic process. In that the junctional complexes developed progressively under repeated stimulation and coupled more and more photosomes, they must represent a route constituted by the ER for the propagation of internal conduction. The dynamics of coupling involve membrane growth, recognition, and transformation on a surprisingly large scale and in a surprisingly short time.     

The Characterization of Scintillons : Bioluminescent particles from the marine dinoflagellate, Gonyaulax polyedra

A new type of biological particle, isolated from the marine dinoflagellate Gonyaulax polyedra, has been partially purified and characterized. When the pH is lowered, the particle emits light in vitro in a fashion closely mimicking the flash of the living cell, and it is referred to as a scintillon (flashing unit). Scintillons are obtained by breaking the cells in buffer at pH 8.2 and purifying by differential and sucrose density gradient centrifugation. The particle has a density of about 1.23 g cc^-1, and activity is quantitatively correlated with the number of crystal-like birhombohedral structures. These have been found to contain guanine, but since the density of authentic guanine is about 1.73 g cc^-1, the scintillon is believed to comprise additional but as yet unidentified components. The properties of the scintillon and the effects of various physical and chemical treatments are described. The reasons for believing that this particle is responsible for the flash of the intact cell are discussed.     

Circadian-clock control of protein synthesis and degradation in Gonyaulax polyedra

In growing cultures of the dinoflagellate, Gonyaulax polyedra, total protein synthesis showed a circadian rhythm with a maximum during the phase of the cycle which corresponded to the previous darktime. The maximum coincided with the maximal phase shift of the glow rhythm caused by lower concentrations of the antibiotic anisomycin (Taylor, W., et al., 1982). J. Comp. Physiol. 148 B, 11–25. The dose reponses of inhibition of protein synthesis correlated well with the phase shifting by anisomycin. The amplitude and level of the total-protein synthesis rhythm increased with the growth rate, indicating that the majority of proteins controlled by the circadian clock were cell cycle-dependent. The degradation rate showed the same circadian rhythm as the synthesis rate. Slight variations in uptake and pool size of amino acids were not responsible for the rhythm in the protein-synthesis rate.     

Effects of Indoleamines and Short Photoperiods on the Encystment of Gonyaulax polyedra

At a temperature of 15^oC, Gonyaulax polyedra responds to short days (light ≤ 10 h) by transition to the stage of a resting cyst. At 20^oC, even an lightdark (LD) cycle of 6:18 is incapable of inducing this process. In otherwise cyst-inducing conditions (15^oC; 10 h of light per day), an interruption of the scotophase by 2 h of light (LDLD 8:2:2:12 or 2:2:8:12) prevented encystment. Cyst induction is, therefore, initiated by a photoperiodic mechanism rather than by light deficiency. In Gonyaulax, photoperiodism may be mediated by the action of indoleamines. Melatonin, which exhibits a circadian rhythmicity in this organism, leads to encystment when given 1 h before lights-off in LD 11:13 at 15^oC, i.e., under otherwise noninducing conditions. Again, at 20^oC, melatonin is inefficient. Some analogues of melatonin, in particular, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine, and, at high concentrations, their respective precursors, serotonin and bufotenin, are capable of inducing cyst formation at 20^oC and in LD 12:12, whereas A'-acetyl-serotonin does not show this effect.     

Effects of Indoleamines and Short Photoperiods on the Encystment of Gonyaulax polyedra

At a temperature of 15^oC, Gonyaulax polyedra responds to short days (light ≤ 10 h) by transition to the stage of a resting cyst. At 20^oC, even an lightdark (LD) cycle of 6:18 is incapable of inducing this process. In otherwise cyst-inducing conditions (15^oC; 10 h of light per day), an interruption of the scotophase by 2 h of light (LDLD 8:2:2:12 or 2:2:8:12) prevented encystment. Cyst induction is, therefore, initiated by a photoperiodic mechanism rather than by light deficiency. In Gonyaulax, photoperiodism may be mediated by the action of indoleamines. Melatonin, which exhibits a circadian rhythmicity in this organism, leads to encystment when given 1 h before lights-off in LD 11:13 at 15^oC, i.e., under otherwise noninducing conditions. Again, at 20^oC, melatonin is inefficient. Some analogues of melatonin, in particular, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine, and, at high concentrations, their respective precursors, serotonin and bufotenin, are capable of inducing cyst formation at 20^oC and in LD 12:12, whereas A'-acetyl-serotonin does not show this effect.     

Circadian rhythms of cell cycle processes in the marine dinoflagellate Gonyaulax polyedra

The circadian expression of several growth properties was examined in the dinoflagellate Gonyaulax polyedra under constant light and light-dark conditions. The cell concentration, mean cell volume and rate of DNA synthesis varied in a circadian rhythm, with the primary maximum of cytokinesis and DNA synthesis at about dawn. High rates of cell mortality also occurred during phases related to events of cytokinesis, and may be important in the expression of the other rhythms and in "red tide" generation. Flow-cytofluorimetric analysis indicated that cells of a population contain either a relatively high or a low amount of DNA, but the proportion of cells in each of these classes and the absolute amount of DNA in each cell varied rhythmically depending on the circadian time. This DNA-distribution pattern was unlike the usual G1-S-G2+M pattern typical of eukaryotic cell populations. Isotopically labelled thymidine, used as a marker of DNA synthesis, was continuously incorporated; but the incorporation rate fluctuated in a regular pattern that repeated each circadian period.     

A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyedra

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.     

The Loss of the Circadian Rhythm in Photosynthesis in an Old Strain of Gonyaulax polyedra

Cultures of Gonyaulax polyedra Stein maintained in the laboratory for 15 to 20 years, including an axenic strain isolated in 1960, have gradually lost the ability to survive in darkness. G. polyedra (70A), isolated in 1970 and maintained in a 12:12 light:dark cycle, now tolerates continuous darkness for a much shorter time than a strain isolated in 1981. I have compared the properties of strain 70A with those of this newer strain (81N), to investigate changes in Gonyaulax with length of time in culture, which may account for poor survival in darkness. When grown in continuous light (13, 12, or 4.5 watts per square meter), strains 70A and 81N have similar growth rates, yields, cell diameters, protein contents, C/N ratios, respiration rates, pigment complements, and photosynthetic rates. When entrained by a light:dark cycle (12L:12D), 70A showed no photosynthesis rhythm, although such a rhythm was formerly present. However, the circadian rhythms in bioluminescence and cell division were normal in both strains. Thus, the circadian clock is apparently still intact in 70A as in 81N. The rate of photosynthesis in strain 70A was constant at a low level, the consequent smaller accumulation of photosynthetic products probably accounting for the limited survival in darkness. The defect in strain 70A may be the loss of a component either directly affecting P_max or necessary for transduction from the circadian clock to photosynthesis.     

A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyedra

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.     

Peridinin as the major biological carotenoid quencher of singlet oxygen in marine algae Gonyaulax polyedra

Carotenoids in light-harvesting proteins and reaction centers increase the overall efficiency of photosynthesis by transferring absorbed light energy to chlorophylls. Peridinin and beta-carotene were isolated from Gonyaulax polyedra in a one-step purification protocol using the preparative circular chromatography (Chromatotron), performed on silica gel under N(2) atmosphere and n-hexane/acetone 8:2 as mobile phase and characterized by extensive (1)H NMR, infrared, and electrospray ionization mass spectrometry analyses. The quenching of singlet molecular oxygen [O(2) ((1)Delta(g))] was evaluated by NIR-emission assays using singlet oxygen generated by sensitization of either perinaphthenone or methylene blue. The NIR-emission assay showed that peridinin quench as singlet oxygen (k(q) = 9.5 x 10(8) M(-1) s(-1)) 5-fold less efficiently than beta-carotene (52 x 10(8) M(-1) s(-1)). A method, based on the use of high-performance liquid chromatography with UV-VIS detection, was then developed for the sensitive quantification of peridinin (55% of total carotenoids) and beta-carotene (4.1% of total carotenoids). Thus, since peridinin is 10-fold more abundant than beta-carotene, it is expected to be the major protector against the deleterious effects of O(2) ((1)Delta(g)) in Gonyaulax polyedra.     

The Action Spectrum for Shifting the Phase of the Rhythm of Luminescence in Gonyaulax polyedra

The action spectrum for changing the phase of the rhythm of luminescence in the marine dinoflagellate Gonyaulax polyedra has been determined. Maxima in effectiveness were found at 475 and 650 mµ. The significance of these findings is discussed.