Molecular/Genetic Analysis of Biological Clocks in Cells

Vanderbilt University, Nashville TN

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Abstract

DESCRIPTION (provided by applicant): Humans and most other organisms manifest circadian (daily) rhythms that are controlled by an endogenous biochemical oscillator. Many cellular processes, including cell division, enzyme activity, and gene expression are timed by this oscillator. These "biological clocks" are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in depressive illness, "jet lag," drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the mechanism of the circadian clockwork and its entrainment may lead to procedures that will be useful in the diagnosis and treatment of disorders which are relevant to sleep, mental health, and pharmacology. One of the fascinations of circadian clocks is that their properties-24 hour time constant (with high precision), temperature compensation, and entrainment-are presently impossible to explain biochemically. Recent breakthroughs in the field of circadian rhythms have identified a number of proteins that appear to act as clock components. Nevertheless, only a little is known about how these components interact functionally with themselves and the environment to generate precise, temperature-compensated 24 hour oscillations that are entrained to the daily cycle. Changes in free calcium levels have been implicated in the entrainment and expression of circadian rhythms. Using new systems and methodological approaches, this project will monitor free calcium levels and fluxes to clarify their role in the entrainment and expression of circadian systems (including its sister phenomenon, photoperiodism). The experimental material will include mammalian cell cultures, mammalian brain slices, and model systems. Luminescence and fluorescence technology will be used to monitor free calcium levels and to track clock gene expression. Free calcium levels will be altered under controlled conditions and concomitant changes in the phase of the circadian clock will be assessed to test hypotheses of the role of calcium fluxes in the entrainment of circadian clocks. Downstream events in the transduction of entraining signals will be studied in mammalian cells (CREB/CBP, cADPR) and model systems (cADPR, ZGT gene) to characterize the phase-resetting pathway.