Daily biological clocks regulate many cellular processes, including cell division, enzyme activity, and gene expression. Psychiatric and medical studies have shown that such circadian rhythms are involved in sleep/activity cycles, mental health, memory, """"""""jet-lag,"""""""" drug tolerance, drug efficacy, and even aging and longevity. A fundamental question in the field concerns the molecular mechanism of circadian oscillators. How are circadian oscillations generated and what components are involved at the cellular and subcellular levels of organization? Our long-term objectives are to understand the cellular and biochemical events that underlie circadian rhythmicity. To this end, the characterization of the circadian system of the unicell Chlamydomonas will be undertaken. This simple cell is particularly useful as a model system for the proposed experimental strategy because it permits the use of mutants in both clock and other physiological functions. It also exhibits circadian rhythmicity of several easily-assayable processes: phototaxis, cell division and adhesion to surfaces. A vigorous search for new examples of clock-controlled gene expression (of mRNA and proteins) will be pursued to provide subjects for studying the mechanism(s) by which the clock pacemaker regulates its overt rhythms. The possible role of cytoplasmic calcium ions in control and/or homeostasis of the clock will be addressed by measuring calcium levels with fluorescent calcium indicators during the circadian cycle and after treatments (e.g., mutation or drugs) which affect circadian frequency. The hypothesis that the photoreceptor for Chlamydomonas clock is a rhodopsin, possibly like the one in vertebrates, will be directly tested by incorporation of rhodopsin analogs into """"""""blind"""""""" mutants. Finally, any membrane proteins which play a role in the clock mechanism will be identified by lectin and/or antibody perturbation of the clock in cell-walless mutants of Chlamydomonas.
