The Biochemistry of Clock Function in Fluctuating Environments

波动环境中时钟功能的生物化学

• 批准号: 10361500
• 负责人: Michael Rust
• 金额: $ 31.92万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10361500
• 关键词: Address; Amino Acid Sequence; Anacystisnidulans; Animal Model; Bacteria; Bacterial Model; Bar Codes; Behavior; Behavioral; Biochemical; Biochemistry; Biological Assay; Biological Clocks; Cell physiology; Cells; Circadian Rhythms; Clock protein; Complex; Coupled; Coupling; Cyanobacterium; DNA sequencing; Data; Data Set; Defect; Dependence; Environment; Feedback; Financial compensation; Functional disorder; Gene Expression; Genes; Genetic Transcription; Goals; Growth; Health; Hour; Human; Impaired health; Impairment; In Vitro; Jet Lag Syndrome; Kinetics; Lead; Libraries; Life; Light; Link; Longevity; Markov Chains; Measurement; Measures; Memory; Metabolic; Metabolism; Modeling; Modernization; Mutagenesis; Mutation; Organism; Output; Pathway interactions; Periodicity; Phase response curves; Phenotype; Phosphorylation; Point Mutation; Process; Property; Proteins; Reaction; Recording of previous events; Research; Rest; Scanning; Signal Pathway; Signal Transduction; Site; System; Temperature; Testing; Time; Tube; Work; base; circadian; circadian pacemaker; fitness; genetic analysis; in vivo; large datasets; mathematical model; mutant; novel; protein complex; reconstitution; response; shift work; stoichiometry; transcriptome sequencing

Project Summary Circadian rhythms are daily oscillations in behavior with a nearly 24-hour period that are generated by an internal biological clock. Across many organisms, health and fitness are impaired when the circadian clock does not appropriately synchronize with the daily cycles in the external environment. It is thus critical to understand how clocks respond to challenging fluctuating environments with intermittent or irregular inputs that are typical of modern life. This problem is conceptually challenging because circadian clocks are complex systems. In general, there is a core oscillator consisting of biochemical circuitry that generates a rhythmic daily signal, and the timing of this rhythm can be adjusted by input signals that communicate information about the environment to the oscillator. However, this oscillator is also embedded in the rest of cellular physiology, and so its response to a changing environment is likely contingent on the status of metabolism and other signaling pathways. We are using the bacterial model organism Synechococcus elongatus to crack the problem of clock-environment interaction because this organism has the remarkable feature that the core oscillator can be reconstituted in vitro using purified proteins. We will thus use a reductionistic approach to build up to an integrated mathematical model of clock function in the intact cell when subject to environmental fluctuations. In Aim 1, we will study the purified test tube oscillator, collecting a large data set of kinetic measurements on the core clock proteins at various temperatures, metabolite concentrations, and protein stoichiometries. Using advanced statistical approaches, we will then constrain a model of elementary reactions to uncover how temperature compensation, metabolic sensing, and entrainment function in the core oscillator. In Aim 2, we will study how the clock shifts in response to environmental fluctuations in the living cell. Here we will use a novel assay to isolate the history-dependence of clock sensitivity that is absent from the core oscillator. We will then use a genetic analysis to find the key pathways used to modulate clock sensitivity in vivo. These data will then be incorporated into a expanded mathematical model that describes the function of the clock in vivo when environmental conditions fluctuate. In Aim 3, we will develop a deep mutagenic scanning approach, to find the clock phenotype and competitive growth defects of 10,000s of point mutations in the clock genes simultaneously. This will not only allow us to discover critical interaction sites on the clock proteins, but also to obtain a comprehensive list of period mutants and mutants that disrupt temperature compensation. Because this assay is based on the transcriptional feedback loops ubiquitous in circadian clocks, it can be generally applied to other clock systems as well.

项目摘要 昼夜节律是行为的每日振荡，具有近24小时的周期，其产生于 体内的生物钟在许多生物体中，当昼夜节律改变时， 时钟与外部环境中的日常周期不同步。因此 对于了解时钟如何响应具有间歇性或间歇性的具有挑战性的波动环境至关重要 现代生活中常见的不规则输入。这个问题在概念上具有挑战性，因为 生物钟是复杂的系统。一般来说，有一个核心振荡器组成的生化 一种产生有节奏的每日信号的电路，这种节奏的时间可以通过输入来调整 将关于环境的信息传达给振荡器的信号。然而，这个振荡器 也嵌入到细胞生理学的其他部分，因此它对变化的环境的反应是 可能取决于代谢和其他信号通路的状态。我们用细菌 模式生物细长聚球藻破解生物钟与环境相互作用问题 因为这种生物体具有核心振荡器可以在体外重建的显著特征， 使用纯化的蛋白质。因此，我们将使用简化的方法来建立一个综合的 当受到环境波动时，完整细胞中时钟功能的数学模型。在 目的1，我们将研究纯化试管振荡器，收集大量的动力学测量数据 在不同温度、代谢物浓度和蛋白质浓度下， 化学计量使用先进的统计方法，我们将约束一个模型的基本 反应，以揭示温度补偿，代谢传感和夹带功能， 核心振荡器在目标2中，我们将研究时钟如何响应环境波动而移动 在活细胞中。在这里，我们将使用一种新的分析方法来分离时钟灵敏度的历史依赖性 这是核心振荡器所没有的。然后我们将使用遗传分析来找到关键途径 用于调节体内的时钟灵敏度。这些数据将被纳入一个扩展的 一个数学模型，描述了生物钟在环境条件下的功能 波动在目标3中，我们将开发一种深度诱变扫描方法，以发现时钟表型 以及时钟基因上一万个点突变的竞争性生长缺陷。这 不仅可以让我们发现时钟蛋白上的关键相互作用位点，而且还可以获得一个 周期突变体和破坏温度补偿的突变体的综合列表。因为 该测定基于生物钟中普遍存在的转录反馈环， 一般也适用于其他时钟系统。