Temperature dependence of cycloheximide-sensitive phase of circadian cycle in Acetabularia mediterranea.

Abstract

The biochemical nature of the circadian regulatory system that controls many cellular activities is still unclear. Recent A variety of cellular activities are regulated by a type of metabolic "clock" which measures time autonomously in intervals of about a day (= circadian) and which can be entrained to other frequencies by environmental cycles of light or temperature (1). The biochemical nature of this circadian regulatory system is still unclear, largely due to the difficulty of distinguishing between its component reactions and the processes which they control. Treatments that alter the period or the phase of a free-running circadian rhythm presumably act directly on the regulatory system, however, and this has been the rationale for the application of a variety of metabolic inhibitors to rhythmic systems (2) as well as for the detection of mutant clock systems (3-5).There are conflicting reports about the effects of inhibitors of protein synthesis on circadian systems. Karakashian and Hastings (6) showed that chloramphenicol had no effect on the phase and period of the glow rhythm in Gonyaulax polyedra, while puromycin blocked the expression of the rhythm and induced small, but reproducible phase delays when it was given as an 8-hr chemical pulse. Sweeney et al. (7) Following the development of a method for continuously monitoring the 02 production of individual Acetabularia cells (9, 10), it was shown that long treatments with cycloheximide altered rhythmic expression in these cells (11) and that short treatments might be affecting phase. The improvement of procedures for describing and comparing the periods and phases of single cell rhythms (12) made it possible to more precisely evaluate the influence of inhibitors on the circadian system of this alga and thereby reexamine the question of whether protein synthesis is necessary for circadian regulation. It was shown recently that an 8-hr exposure to cycloheximide (0.1 jg/ml) or puromycin (50 ,ug/ml) delays the phase of the photosynthesis rhythm 6-14 hr, providing the drug is present during a discrete part of a cell's circadian cycle (13). Similar treatments with chloramphenicol (100 ,g/ml) had no effect on phase. These results, obtained for cells kept at 250, indicate that one or more proteins synthesized on 80S ribosomes participate in the mechanism for circadian control of photosynthesis in Acetabularia.In the present contribution it will be shown that the phase of cycloheximide sensitivity is somewhat longer and occurs about 8 hr later in the circadian cycle, when cells are investigated at 200 rather than 250. As will be discussed, this observation favors the interpretation that 806 protein synthesis, while necessary for the circadian regulation of photosynthesis, does not itself generate the period of the rhythm.

On the role of gene expression in the circadian oscillator mechanism

“…In contrast to other hypotheses, such a sequentiality is built-in in the model by , who suggest the protein synthesis step with Qio>l being followed by a step with Qio<l, e.g., integration of protein into the membrane. This concept would be consistent with the surprising finding of large differences in the phases of maximal sensitivity to cycloheximide at different temperatures (Karakashian and Schweiger, 1976c;Schweiger and Karakashian, 1977). On the other hand, the extent of this temperature effect, resulting in phase positions about 180° apart upon temperature changes of not more than 5°C, certainly exceeds any expectations.…”

Neuronal Circadian Rhythm: Phase Shifting by a Protein Synthesis Inhibitor

“…Interestingly, the reversal point for phase responses to light occurs during the minimum CAP frequency [(12), projected night]. In Acetabularia (17) and Aplysia the delays in phase caused by inhibitors of 80S protein synthesis occurred at similar times in the circadian cycles (02 production at 20°C or CAP activity at 15°C). This common action on such diverse organisms suggests a common mode of action for this class of inhibitors on circadian rhythms generally.…”

Identification of a high molecular weight polypeptide that may be part of the circadian clockwork in Acetabularia

“…By turnover, the amount of this essential polypeptide in the membrane falls below a critical threshold, thus restoring the synthesis of the essential protein and enabling a new cycle to begin. In accordance with this model, the synthesis of the essential protein should exhibit oscillations in the absence of any "Zeitgeber"-in other words, under constant conditions.The coupled translation-membrane model is in agreement with a number offeatures of circadian rhythms, including the cycle-dependent, phase-shifting effect of cycloheximide that has been observed not only for the photosynthesis rhythm in the unicellular green alga Acetabularia (11,12), but also for a membrane potential in the Aplysia eye (13), for a bioluminescence rhythm in Gonyaulax (14,15), for a conidiation rhythm in Neurospora (16), for a chloroplast-migration rhythm in Acetabularia (unpublished data), and for a membrane-potential rhythm in the same organism (unpublished data). Results from genetic experiments in Drosophila (17)(18)(19)(20) suggest that the per gene has a critical role in the expression of a circadian rhythm.…”