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Indicative of the importance of protein phosphorylation in the core circadian clock mechanism, chronically applied inhibitors of both protein kinases and phosphoprotein phosphatases have significant effects on the period, phase, and light-dependent regulation of circadian rhythms in the dinoflagellate Lingulodinium polyedrum. This study was aimed at identifying the presence of the affected phosphatase(s). Dephosphorylation of a PP1/PP2A-specific substrate by L. polyedrum extracts was inhibited by okadaic acid only at concentrations greater than 100nM, as in vivo, by mammalian inhibitor-2 (I-2), and by an endogenous inhibitor with properties similar to I-2, indicating that a type-1 protein phosphatase (PP1) was predominant. AcDNA encoding a highly conserved PP1 was isolated, the 1st such signaling molecule identified in dinoflagellates. Antisera specific for this type of phosphatase recognized a 34 kDa protein in L. polyedrum extract, this being the same size as the PP1 encoded by the isolated cDNA. These findings are consistent with the suggestion that the L. polyedrum PP1 may be a part of the clock mechanism in this species. [ABSTRACT FROM AUTHOR]

Presents a lecture on biological rhythms. People involved in studies on circadian biology; Works on the culture of the marine dinoflagellate Gonyaulax polyedra; Expedition on the research vessel Alpha Helix.

Presents information on a study which characterizes the unusual action of the protein kinase inhibitor staurosporine on the Gonyaulax circadian system. Materials and methods; Results; Discussion.

Describes that phase shifts in response to light pulses differ for two different rhythms in the unicellular alga Gonyaulax polyhedra. Dinoflagellates; Circadian oscillator; Phase response curve; Spectral effects; Photoperiod.

Constant red light (RR) influences the Gonyaulax clock in several ways: (1) Phase resetting by white or blue light pulses is stronger under background RR than in constant white light (WW); (2) frequency of the rhythm is less in RR than in WW; and (3) the amplitude of the spontaneous flashing rhythm is greater in RR than in WW. The phase response curve (PRC) to 4-hr white or blue light pulses is of high amplitude (Type 0) for cells in RR, but is of lower amplitude (Type 1) for cells in WW. In all cases, the PRC is highly asymmetrical: The magnitude of advance phase resetting is far higher than that of delay resetting. Consistent with this PRC, Gonyaulax cells in RR (free-running period greater than 24 hr) will entrain to T cycles of between 21 and 26.5 hr. The bioluminescence rhythms exhibit "masking" by blue light pulses while entrained to these T cycles. The fluence response of phase resetting to light-pulse intensity is not linear or logarithmic—rather, it is discontinuous. This feature is consistent with a limit cycle interpretation of Type 0 resetting of circadian clocks. Light pulses that cause large phase shifts also shorten the subsequent free-running period. The phase angle difference between the clock and the previous LD cycle is within 2 hr of the same phase between 16°C and 25°C, as determined from the light PRCs at various temperatures. Several drugs that inhibit mitochondria and/or electron transport will partially inhibit the phase shift by light. [ABSTRACT FROM PUBLISHER]

Populations of Paramecium bursaria cells display a circadian rhythm of photoac cumulation. Both Chlorella-containing cells and Chlorella-free cells exhibit this rhythm. Sev eral other species of Paramecium do not express rhythmic photoaccumulation when tested under the same conditions. In P. bursaria, photoaccumulation rhythms persist in continuous conditions (constant temperature and either continuous light or continuous darkness). The period of this rhythm is "temperature-compensated," with a Q10 of 1.10-1.12. The rhythm can be reset by pulses of light or darkness in a phase-dependent manner. Therefore, an endoge nous circadian oscillator controls photoaccumulation behavior in P. bursaria. [ABSTRACT FROM PUBLISHER]

Like other flagellates, Gonyaulax polyedra exhibits diurnal vertical migration and pattern formation. Shape and size of the aggregations depend on container type, light inten sity, 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. [ABSTRACT FROM PUBLISHER]

A size filtration method to synchronize cultures of the dinoflagellate Gonyaulax polyedra to the beginning of the G1 phase has been developed. This technique selects newly born cells by two sequential filtrations, based on the fact that cell division is restricted to the beginning of the day, so that a decrease in cell volume occurs at this time. The fraction of synchronized cells immediately after the second filtration is about 90%; the procedures do not alter the free-running period or phase of glow rhythm, and the selected cells divide again in a few days. Applying this method, we have found that the generation times of this species in a light-dark cycle (LD 12:12) are indeed quantized to multiples of 24 hr, but are variable from generation to generation. [ABSTRACT FROM PUBLISHER]

We describe a method whereby the effect of protein synthesis inhibitors upon protein synthesis in Gonyaulax cultures may be reliably measured. Using this method, we found that protein synthesis inhibition and clock resetting were correlated, but that the correlation was not as close as has been reported in other systems. The effect of the inhibitors anisomycin and cycloheximide upon phase shifting of the circadian clock was a function of the illumination and temperature conditions to which the cells were subjected, but these factors did not appear to influence the inhibition of protein synthesis by these drugs. Cellular protein synthesis did not recover immediately from the inhibitors' effects; depending upon the previous concentration of the inhibitor, translational recovery from the drugs may require hours. This observation has important implications for the analysis of any phase response curve when the stimulus is a chemical. [ABSTRACT FROM PUBLISHER]

During measurements of the circadian (approximately 24-hr) rhythms of spontaneous bioluminescence in the marine dinoflagellate Gonyaulax polyedra, the individual cultures in vials were shielded from otherwise constant dim light for 1-3 min every 20-60 min by a photomultiplier housing that was moved from vial to vial. The high-frequency dark pulses caused a small but consistent shortening of the free-running circadian period, but there was no indication that the dark pulses caused entrainment. Hardware and software components of the microcomputer-controlled data collection system are described. A microcomputer controlled the movement of the photomultiplier and acquired the data via an analog-to-digital converter. The algorithms distinguished and separately recorded background glow, intermittent flashes, and total light from populations ranging in number from 103 to 105 cells in volumes from 1 to 10 ml. Fast video display techniques allowed continuous on-line viewing of incoming data, together with a display of the data recorded over the preceding day or two. Detection of mechanical and software errors coupled with recovery systems maintained high reliability of data collection. [ABSTRACT FROM PUBLISHER]