Molecular and Cellular Studies of Circadian Rhythms

我们一直在研究以果蝇为模型系统的昼夜行为节律的分子控制。最初在苍蝇身上发现的基因同源物，现在已经与脊椎动物（包括鱼、青蛙、老鼠和人类）的节律行为和生理控制联系起来。果蝇生物钟的一个核心组成部分是一个反馈回路，其中两个生物钟蛋白PERIOD （PER）和TIMELESS （TIM）抑制它们自己的转录。这种反馈的时间延迟促进了振荡性基因表达。我们最近发现了控制这种延迟的新的细胞特征。另外的研究发现了影响生物钟周期性的新基因和蛋白质。在本提案中，我们将研究以下内容：(1)PER和TIM在细胞质中相互作用时似乎发生了物理修饰，从而允许它们随后独立的核积累。我们将识别和表征与这种调节相关的这些蛋白质的修饰。(2)我们将确定新发现的PER/TIM细胞质间隔计时器是否有助于昼夜节律钟的温度补偿。(3)我们将对培养细胞中调节PER和TIM定时核积累的新基因和蛋白进行高通量筛选。(4)机车

We have been studying the molecular control of circadian behavioral rhythms using Drosophila as a model system. Homologues of genes initially characterized in the fly, have now been linked to the control of rhythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. A central component of the fly clock is a feedback circuit in which two clock proteins, PERIOD (PER) and TIMELESS (TIM), repress their own transcription. Temporal delays in this feedback promote oscillatory gene expression. We have recently discovered novel cellular features controlling one such delay. Additional studies have identifed new genes and proteins affecting periodicity of the circadian clock. In this proposal we will examine the following: (1) PER and TIM appear to be physically modified in response to their interaction in the cytoplasm, allowing their subsequent, independent nuclear accumulation. We will identify and characterized modifications of these proteins that are associated with this regulation. (2) We will determine whether a newly discovered, PER/TIM cytoplasmic interval timer contributes to temperature compensation of the circadian clock. (3) We will conduct a high-throughput screen for new genes and proteins regulating the timed nuclear accumulation of PER and TIM in cultured cells. (4) A locomotor activity screen involving several hundred transgenic RNAi stocks has shown that reduction of a specific karyopherin substantially lengthens the period of the fly clock. The molecular pathway underlying this protein's contribution to rhythmicity will be explored in flies and S2 cells. (5) Cryptochrome (CRY) has a key role in the light-dependent degradation of TIM. We have produced new mutations that affect physical interactions of the CRY C-terminal tail with the CRY photolyase homology domain (PHD). Preliminary studies indicate that some of these mutations alter CRY stability only on exposure to light. We will determine whether light induces dissociation of the CRY C-terminal tail and the PHD. RELEVANCE (See instructions): Candidate gene approaches, originating in the forward genetic screens of Drosophila, allowed mutant orthologs of human PERIOD protein and casein kinase 1 to be connected to inborn errors of sleep. The early functional studies of these genes and proteins in Drosophila have also been used as the basis for exploring specific mechanisms underlying aberrant patterns of human sleep. We believe our proposed genetic, biophysical, and biochemical studies of Drosophila's circadian clock will continue to reveal new principles of organization and function that promote an understanding of human circadian rhythms.