A circadian rhythm of population behavior in Gonyaulax polyedra

Like other flagellates, Gonyaulax polyedra exhibits diurnal vertical migration and pattern formation. Shape and size of the aggregations depend on container type, light intensity, and cell density. In Petri dishes, cells form oval "swarms"; within these, cells move downward in the highly dense center and rise up at the periphery. We have investigated the daily rhythm of this swarming activity in Petri dishes illuminated from the side, using time-lapse video recordings. At night, a "lawn" of cells forms at the bottom of the dish toward the light source (independent of light intensity). Before dawn, cells rise toward the surface and aggregate in swarms. The daily vertical migration occurs independent of light direction and intensity. The diurnal swarms, however, form every day at the same location within the dish, at a distance from the light that depends on light intensity, indicating a self-selection of light intensity. In constant light and temperature and with negligible vertical nutrient differences, all aspects of the rhythm continue to oscillate for up to 3 weeks, when the rhythm of the population becomes desynchronized. Under cycles of bright white-dim red light (WR), cell entrain to WR 10:10 but free run in WR 8:8 and shorter cycles, showing relative coordination (von Holst, 1939) to the driving light cycle. They also entrain to the 24-hr multiple of WR 6:6. Under nonentrained conditions, swarming activity is still influenced by light changes, and in spite of the apparent free run, the phasing of the averaged activity varies systematically with different T-cycle frequencies.     

The complex circadian system of Gonyaulax polyedra

Circadian oscillations are a fundamental biological property from bacteria to humans. The molecular mechanisms which produce a ca 24-h rhythmicity are still unknown but it has become clear that they are part of the biochemical machinery of the single cell. The cellular circadian system can be favorably studied in single-cell organisms such as the dinoflagellate Gonyaulax polyedra . The complexity of this circadian model system, which consists of at least two circadian oscillators, receives light via two input systems with different spectral sensitivities, and has several feed–back loops between the central oscillator(s) and the environment, is described here.     

Phase of the circadian clock is accurately transferred from mother to daughter cells in the dinoflagellate Gonyaulax polyedra.

In the first cycle following transfer from a 12 h light-12 h dark cycle (LD12:12) to constant darkness (DD), the standard deviation in circadian phase among individual clocks in populations of Gonyaulax polyedra is approximately 60 min. When a culture is transferred to constant light conditions (LL) from an LD 12:12 cycle, the standard deviation increases in the first 2-3 d, but then remains unchanged, suggesting a lack of observable desynchronization in LL after the transient period. The synchrony in a cell population is preserved even after several cell divisions. The results indicate that variations in period among cells are small, that the period of an individual clock does not fluctuate randomly from day to day, and that the circadian phase of a mother cell is faithfully passed to the clocks of the daughter cells.     

The s phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra

Cloned cultures of the dinoflagellate Gonyaulax polyedra grown in a 12-h light-12-h dark cycle (LD 12:12) were synchronized to the beginning of G1 by a two sequential filtration technique. After the second filtration, with the cultures growing in LD 12:12, not many cells had divided after 1 day, but approximately half underwent cell division after 2 days. Flow cytometric measurements of the cells revealed that there is one unique S phase starting about 12 h prior to cell division and lasting for less than 4 h. A majority of cells in cultures synchronized in the same way but maintained in continuous light (LL) after filtration also divided synchronously after 2 days. Just as for the cultures in LD 12:12, those in LL have a similar discrete DNA synthesis phase prior to division. It is concluded that the circadian control of cell division acts before the S phase, giving rise to a discontinuous DNA synthesis phased by the circadian clock.     

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.     

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.     

Effects of aldehydes and alcohols on protein synthesis in the dinoflagellate, Gonyaulax polyedra: relation to phase-shifting potency in the circadian oscillation

In Gonyaulax polyedra, protein synthesis was measured in vivo and in an in vitro translating system. Aldehydes applied in concentrations by which the circadian oscillator is phase-shifted inhibited protein synthesis considerably, both in vivo and in vitro. The efficiency of the drugs decreased with increasing chain length. Alcohols, though given in much higher concentrations, showed only weak effects. Therefore, the phase-shifting potency of aldehydes can be explained by a general mechanism influencing the circadian oscillator, namely through inhibition of protein synthesis, whereas this is obviously not the case in alcohols.     

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.     

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.     

A circadian rhythm of the luciferin binding protein fromGonyaulax polyedra

Summary It has previously been shown that a protein extracted fromGonyaulax polyedra strongly and specifically binds luciferin, the substrate of the bioluminescent reaction. This binding is markedly dependent on pH with tight binding at pH 8.0 and almost no binding at pH 6.5, as measured by two independent methods. A procedure for the determination of the dissociation constant (K_d) of the luciferin binding protein (LBP) is presented, and K_d is estimated to be7×10^–9 M at pH 8.0, assuming an overall quantum yield of 0.1 for the bioluminescent reaction. With cells grown in a 12 h light — 12 h dark cycle, 5 to 10 times more LBP activity can be extracted from dark phase cells than from light phase cells. This rhythm persists in a circadian fashion in cultures maintained in constant dim light.Supported in part by a grant from the National Institutes of Health to J.W.H. (GM 19536)     

Reassessing the role of a 3'-UTR-binding translational inhibitor in regulation of circadian bioluminescence rhythm in the dinoflagellate Gonyaulax

The nightly bioluminescence of the dinoflagellate Gonyaulax is a circadian rhythm caused by the presence in cells of specialized bioluminescent organelles, termed scintillons, containing the reaction catalyst luciferase, the substrate luciferin and a luciferin-binding protein (LBP). LBP levels increase at the start of the night phase because of increased protein synthesis rates in vivo, and this regulation has been ascribed to circadian binding of an inhibitory protein factor binding to the 3' untranslated region (UTR) of lbp mRNA at times when LBP is not normally synthesized. To purify and characterize the binding factor, the electrophoretic mobility shift assays and UV crosslinking experiments used to first characterize the factor were repeated. However, neither these protocols nor binding to biotinylated RNA probes confirmed the presence of a specific circadian RNA-binding protein. Furthermore, neither RNA probe screening of a cDNA library expressed in bacteria nor three-hybrid assays in yeast were successful in isolating a cDNA encoding a protein able to bind specifically to the lbp 3'UTR. Taken together, these results suggest that alternative mechanisms for regulating lbp translation should now be examined.     

Corrigendum - The structure and organization of the luciferase gene in the photosynthetic dinoflagellate Gonyaulax polyedra

Plant Molecular Biology -     

The chemical mimicking of the mechanical stimulation, photoinhibition, and recovery from photoinhibition of bioluminescence in marine dinoflagellate, Gonyaulax polyedra

Mechanically stimulable bioluminescence and photoinhibition of sensitivity to mechanical stimulation in the marine dinoflagellate Gonyaulax polyedra can be mimicked by a number of cations, proportional to the logarithm of their external concentrations. The data are consistent with mechanical stimulability as a membrane depolarization resulting in an increase in H+ ions at bioluminescence sites and with photoinhibition as a hyperpolarization of the cell membrane.     

Inhibitors of serine/threonine phosphoprotein phosphatases alter circadian properties in Gonyaulax polyedra.

Protein serine/threonine phosphatases were implicated in the regulation of circadian rhythmicity in the marine dinoflagellate Gonyaulax polyedra based on the effects of three inhibitors specific for protein phosphatases 1 and 2A (okadaic acid, calyculin A, and cantharidin). Chronic exposure to okadaic acid resulted in a significant period lengthening, as measured by the bioluminescent glow rhythm, whereas cantharidin and calyculin A caused large phase delays but no persistent effect on period. Short pulses of the phosphatase inhibitors resulted in phase delays that were greatest near subjective dawn. Unlike 6-dimethylaminopurine, a protein kinase inhibitor, okadaic acid, calyculin A, and cantharidin did not block light-induced phase shifts. The inhibitors tested also increased radiolabeled phosphate incorporation into Gonyaulax proteins in vivo and blocked protein phosphatase 1 and 2A activities in Gonyaulax extracts. This study indicates that protein dephosphorylation catalyzed by protein serine/threonine phosphatases is necessary for proper functioning of the circadian system.     

Extracellular pH is under circadian control in Gonyaulax polyedra and forms a metabolic feedback loop

This study investigates the relationship between the circadian clock and metabolism based on recordings of the extracellular pH in cultures of the marine dinoflagellate, Gonyaulax polyedra. In light-dark cycles, pH of the medium rises during the light phase and declines in the dark. The amplitude of this pH-rhythm correlates with light intensity, indicating photosynthesis (and respiration) as the driving force. The recorded extracellular pH changes probably reflect the need to control intracellular pH in spite of pH-modifying reactions. The daily pH-changes are under control of the circadian clock because they continue to oscillate with a circa-24 h period in constant light, albeit with a smaller amplitude. Similar to other circadian output rhythms, the pH rhythm depends (amplitude and phase) on nitrate levels in the medium. Both the bioluminescence and the pH rhythm can also be shifted by extracellular pH-changes although Gonyaulax is rarely exposed to significant pH changes in its marine ecosystems (except for highly dense algal blooms). Because intracellular proton levels are both affecting circadian input and output they form a feedback loop with the Gonyaulax circadian system indicating complex interactions between metabolism and the circadian clock.