Freeze-fracture studies of the thecal membranes of Gonyaulax polyedra: circadian changes in the particles of one membrane face.

Intramembrane faces were visualized in the marine dinoflagellate Gonyaulax polyedra by the freeze-fracture technique, in order to test a prediction of a membrane model for circadian oscillations--i.e;, that membrane particle distribution and size change with time in the circadian cycle. Cells from each of four cell suspensions in continuous light (500 1x, 20-21 degrees C) were frozen, without fixation or cryoprotection, at four circadian times in a cycle. This paper reports findings concerning the membranes associated with the theca, particularly the cytoplasmic membrane and the membrane of the large peripheral vesicle. While the number and size distribution of the particles of the PF face of the cytoplasmic membrane were constant with time, those of the EF face of the peripheral vesicle doubled in number at 18 h circadian time as compared with 06 h. Particles of the 120-A size class, in particular, were more numerous at 12 and 18 h circadian time than at 00 and 06 h. While the finding does not provide definitive confirmation of the membrane hypothesis for circadian rhythms, it is consistent with this model. It is suggested that the peripheral vesicle may be the site of bioluminescence in Gonyaulax.

Pigment protein complex from gonyaulax.

A water-soluble peridinin-chlorophyll-protein complex from the dinoflagellate Gonyaulax polyedra was found to have a molecular weight of about 38,000. The complex could be disrupted by digestion with proteolytic enzymes. No electron transfer was observed when the complex was irradiated.

The circadian rhythm in photosynthesis in Acetabularia in the presence of actinomycin D, puromycin, and chloramphenicol.

Anucleate Acetabularia crenulata shows a circadian rhythm in photosynthesis. In this study, an oxygen electrode was employed to measure this photosynthetic rhythm in the presence and absence of the inhibitors, actinomycin D, chloramphenicol, and puromycin. High concentrations of the inhibitors were used: actinomycin D, 20-40 micrograms ml-1; puromycin, 30 and 100 micrograms ml-1; and chloramphenicol, 250 micrograms ml-1. The effectiveness of these inhibitors on protein synthesis was also measured under the same conditions used for the determination of rhythmicity. In spite of large effects of all three inhibitors on the incorporation of 14C leucine, no effect on the period or the phase of the photosynthetic rhythm was observed. The higher concentration of puromycin and chloramphenicol produced toxic effects which were expressed as a reduction in the amount of photosynthesis, but rhythmicity was still apparent. After 3 or 4 days' exposure to actinomycin, Acetabularia became resistant to its effect. Recovery was also observed in the ability to incorporate leucine. The implications of these results for theories of the basic oscillator responsible for circadian rhythmicity are discussed.

CULTURE STUDIES ON THE MARINE GREEN ALGA HALICYSTIS PARVULA-DERBESIA TENUISSIMA. III. CONTROL OF GAMETE FORMATION BY AN ENDOGENOUS RHYTHM(1) (2).

Evidence is presented for an endogenous rhythm which controls gamete formation in the coenocytic gametophytic stage of the marine alga Derbesia tenuissima (De Notaris) Crouan fr. (Chlorophyceae), formerly known as Halicystis parvula. The rhythm is present under conditions of constant light and temperature, or in alternating light and darkness (LD 12 :12 or LD 8 : 8). Under controlled conditions in the laboratory, the basic period of this rhythm is 4-5 days. The period is affected very little by temperature or light intensity. Gametangia may appear only at every other cycle of the underlying rhythm at approximately 8-day intervals, or even every third cycle at intervals of about 12 days. The observation that the interval between the appearance of new gametangia does not vary continuously when light or temperature is varied but tends to be a multiple of 4 days is the strongest evidence available that a true endogenous rhythm with a period of about 4 days is present in Derbesia. Evidence is presented that circadian rhythmicity does not play a part in the timing of the initiation of gametangia in this organism.

Interaction of the Circadian Cycle with the Cell Cycle in Pyrocystis fusiformis.

Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G(1), DNA synthesis, S, the second growth stage, G(2), mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4', 6-diamidino-2-phenylindole dichloride.Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light-dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G(q), an hypothesized side loop from G(1). All morphological stages, not only division, appear to be phased by the circadian clock.

Bright light does not immediately stop the circadian clock of gonyaulax.

Circadian rhythms in acid-stimulated bioluminescence and cell division are observed for at least 16 days in bright continuous light (4.5 milliwatts per square centimeter or 20,000 lux). The photosynthesis rhythm also fails to stop immediately upon transfer of cell suspensions to bright light. After about 4 weeks under these conditions, all rhythms were observed to damp out. In cells transferred from bright light to continuous darkness, the rhythms were reset to about circadian hour 12 to 14, the phase of the beginning of a normal night.

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.

The Potassium Content of Gonyaulax polyedra and Phase Changes in the Circadian Rhythm of Stimulated Bioluminescence by Short Exposures to Ethanol and Valinomycin.

A circadian rhythm in the intracellular level of K(+) in Gonyaulax polyedra is reported. When axenic cultures of Gonyaulax in continuous light (60-75 fot candles) are exposed for 4 hours to 0.1 or 0.2% ethanol, the subsequent free-running rhythm in stimulated bioluminescence is phase-shifted, the amount and direction of the shift being dependent on the time in the circadian cycle when cells are treated. The phase-response curve for ethanol closely resembles that for light in similarly maintained cells. When valinomycin (0.1 or 0.2 mug ml(-1)) is present in addition to ethanol, the phase of the bioluminescence rhythm is returned to that of an untreated cell suspension. Valinomycin thus negates the effect of ethanol on phase. The intracellular K(+) level immediately after treatment of a cell suspension for 4 hours with ethanol (0.1%) is about half that of untreated cells. If valinomycin (0.1 mug ml(-1)) is also present during the 4-hour treatment, the intracellular K(+) is only slightly lower than in untreated cells. Increasing the external concentration of K(+) or Na(+) for 4 hours has no effect on the rhythm of stimulated bioluminescence. These results are interpreted as support for the hypothesis that the mechanism by which circadian oscillations are generated involves changes in membrane properties.

Nitrate deficiency shortens the circadian period in gonyaulax.

The circadian rhythms in bioluminescence and photosynthesis in Gonyaulax polyedra suspended in unsupplemented sea water have been compared to the same rhythms in f/2, an enriched seawater medium. Cells suspended in sea water for 2 days in continuous light (450 microwatts per square centimeter) showed significantly shorter circadian periods and lower amplitudes than did cells in f/2 medium (a period of 22.2 hours as compared to 23.5 hours). Both period and amplitude changes could be completely reversed by the addition of nitrate at one-fourth or more of the concentration in f/2 medium (0.88 millimolar). The addition to autoclaved seawater of phosphate, vitamins, minerals, or soil extract in concentrations present in f/2 medium had no effect. Thus, the shortening of the circadian period is the consequence of reduced nitrogen supply. Since both the rhythms in bioluminescence and photosynthesis showed similarly shortened circadian periods and lower amplitudes, it is probable that the depletion of nitrate directly affects the circadian clock.

Characterization of Photosynthetic Rhythms in Marine Dinoflagellates: II. Photosynthesis-Irradiance Curves and in Vivo Chlorophyll a Fluorescence.

Using data from light-dark cultures of Gonyaulax polyedra entrained to a 24-hour cycle, whole cell absorption curves and photosynthesis-irradiance curves were constructed for various circadian times. While whole cell absorbance and half-saturation constants of photosynthesis showed no statistical difference that could be directly related to the photosynthetic rhythm, the initial slope of the photosynthesis-irradiance curve was a time-dependent parameter which altered in direct proportion to the change in photosynthetic capacity. The results indicated a temporal change in the relative quantum yield of photosynthesis, and the circadian rhythmicity of light-limited photosynthesis was established under constant conditions. Circadian rhythmicity was detected in room temperature chlorophyll fluorescence yield. Low temperature fluorescence kinetics also showed fluctuations. The results suggest that regulation of photosynthesis by the biological clock of Gonyaulax may be mediated through the membrane-bound light reactions and a partial explanation of the underlying mechanism is proposed.

The preparation and characterization of Gonyaulax spheroplasts.

Viable spheroplasts of a marine dinoflagellate, Gonyaulax polyedra, have been prepared for the first time. This simple and rapid procedure results in a yield of over 95% intact spheroplasts. Utilizing this technique, many studies of the cell-wall-free form of this dinoflagellate are now possible.

Kinetics of the cycloheximide-induced phase changes in the biological clock in Gonyaulax.

Cycloheximide, an inhibitor of protein synthesis on cytoplasmic ribosomes in eukaryotes, is shown to shift the phase of the circadian rhythm in stimulated bioluminescence in the marine dinoflagellate Gonyaulax polyedra. Kinetic analysis of the phase changes shows that this effect may be subdivided into two distinctly different and well-separated parts. The first (early) phase change occurs with 15-min exposure to cycloheximide and is saturated at low concentrations ( approximately 10 nM). The second (late) phase changes requires about 150 min of exposure to cycloheximide and is saturated at 0.36 muM cycloheximide. Twenty-times-higher concentrations cause no further phase changes. The magnitudes of both early and late phase changes depend on the time of day when the cells are exposed to cycloheximide. Early phase changes vary from 5 hr advance at circadian time (CT) 20 to 1 hr delay at CT 12; late phase changes are larger, the maximal advance being 12 hr at CT 16 and the greatest delay, 10 hr at CT 14. It is proposed that the early phase changes are caused by alterations in the ion distribution across membranes as a consequence of the permeation of cycloheximide. Late phase changes may be the result of inhibition of protein synthesis.The phase response curve for the late phase change is identical to that obtained with saturating light pulses in otherwise constant darkness in Gonyaulax. Maximal phase changes drive the clock into the part of the circadian cycle between CTs 4 and 9. Perturbations in this part of the circadian cycle are without effect on phase. Incubation of Gonyaulax with cycloheximide for a critical duration at a critical time induces arhythmicity, but longer exposures to the inhibitor at the same time do not. This observation suggests the existence of a singularity in the circadian clock of Gonyaulax.

Sensitivity to stimulation, a component of the circadian rhythm in luminescence in gonyaulax.

A new method for the stimulation of bioluminescence in the dinoflagellate Gonyaulax polyedra is described. With this technique, in which cells flow through a capillary coil, it is possible to graduate the intensity of the stimulus by varying the flow rate. In continuous darkness, the threshold stimulus for cells in the middle of the day phase is greater than that for cells in the middle of the night phase. Some evidence suggests heterogeneity of sensitivity to stimulation among either cells or individual luminescent sources within a cell. At stimulus intensities much above threshold, the luminescence of both day- and night-phase cells is proportional to the number of cells within the capillary coil. Night-phase cells emit about 14 times as much light as do day-phase cells in continuous darkness.Single bioluminescent flashes from cells were recorded with a high speed camera. No significant difference in flash kinetics was found between cells in the day and the night phase in continuous darkness. Cells in the night phase emit a flash three to five times brighter than that from day-phase cells. About twice as many flashes are recorded in a given time from a population of night-phase cells.The activity of both luciferin and luciferase have been shown to vary rhythmically. The differences in threshold and number of flahses are evidence for a second component of the circadian rhythm in luminescence, a rhythm in sensitivity to stimulation.

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis.

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg(2+) concentration is 3 mm. Heavy metal ions (Cu(2+), Hg(2+), Ni(2+), Zn(2+)), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO(3) (-) was also the same (16 to 19 mm).

The ultrastructural localization of luciferase in three bioluminescent dinoflagellates, two species of Pyrocystis, and Noctiluca, using anti-luciferase and immunogold labelling.

In order to discover the intracellular location of luciferase in dinoflagellates, sections from a number of species were treated with a polyclonal anti-luciferase and the bound antibody was visualized at the electron-microscope level by indirect immunogold labelling. In two species of Pyrocystis and in Noctiluca, as in Gonyaulax, antibody became bound to dense vesicles, which correspond in size and position to light-emitting bodies detected in previous work. These vesicles resemble microsomes, are bounded by a single membrane and sometimes project into the vacuole. Unexpectedly, the trichocysts of Gonyaulax and Noctiluca and the related mucocysts of Pyrocystis also bound the antibody. This cross-reaction seems quite independent of bioluminescence, since the trichocysts of the non-luminous Cachonina also reacted positively. The possibility is discussed that a protein, different from luciferase but having some antigenic similarity, is present in trichocysts and related organelles.