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: 10586111
• 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/10586111
• 关键词: Address; Amino Acid Sequence; Anacystisnidulans; Bacteria; Bacterial Model; Bar Codes; Behavior; Behavioral; Biochemical; Biochemistry; Biological Assay; Biological Clocks; Cell physiology; Cells; Circadian Rhythms; Clock protein; Communication; Compensation; Complex; Coupled; Coupling; Cyanobacterium; DNA sequencing; Darkness; Data; Data Set; Defect; Dependence; Environment; Feedback; Functional disorder; Gene Expression; Genes; Genetic Transcription; Goals; Growth; Health; Hour; Human; Impaired health; Impairment; In Vitro; Jet Lag Syndrome; Kinetics; 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; circadian; circadian pacemaker; fitness; genetic analysis; in vivo; large datasets; mathematical model; model organism; mutant; novel; protein complex; protein purification; 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.

项目概要 昼夜节律是近 24 小时周期内的每日行为波动，由以下因素产生： 内部生物钟。在许多生物体中，当昼夜节律改变时，健康和健身就会受到损害。 时钟与外部环境中的日常周期不适当同步。正是如此 了解时钟如何应对具有间歇性或间歇性波动的挑战性环境至关重要 现代生活中典型的不规则输入。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，有一个由生化组成的核心振荡器 产生有节奏的每日信号的电路，并且可以通过输入调整该节奏的时间 将有关环境的信息传递给振荡器的信号。然而，这个振荡器 也嵌入到细胞生理学的其余部分中，因此它对不断变化的环境的反应是 可能取决于新陈代谢和其他信号通路的状态。我们正在使用细菌 模型生物细长聚球藻破解时钟-环境相互作用问题 因为这种生物体有一个显着的特点，就是核心振荡器可以在体外重构 使用纯化的蛋白质。因此，我们将使用简化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 目标1，我们将研究纯化的试管振荡器，收集大量的动力学测量数据集 不同温度、代谢物浓度和蛋白质下的核心时钟蛋白 化学计量。使用先进的统计方法，我们将约束基本模型 反应以揭示温度补偿、代谢传感和夹带如何在 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变化 在活细胞中。在这里，我们将使用一种新颖的测定法来分离时钟灵敏度的历史依赖性 这是核心振荡器所不存在的。然后我们将使用遗传分析来找到关键途径 用于调节体内时钟敏感性。然后这些数据将被合并到一个扩展的 描述生物钟在环境条件下的功能的数学模型 波动。在目标 3 中，我们将开发一种深度诱变扫描方法，以找到时钟表型 以及时钟基因中数万个点突变同时导致的竞争性生长缺陷。这 不仅能让我们发现时钟蛋白上的关键相互作用位点，还能获得 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，它可以 通常也适用于其他时钟系统。

项目成果

KidA, a multi-PAS domain protein, tunes the period of the cyanobacterial circadian oscillator.

KidA 是一种多 PAS 结构域蛋白，可调节蓝藻昼夜节律振荡器的周期。

• 发表时间: 2022-09-13
• 影响因子: 11.1
• 作者: Kim, Soo Ji;Chi, Chris;Pattanayak, Gopal;Dinner, Aaron R;Rust, Michael J
• 通讯作者: Rust, Michael J

The circadian clock ensures successful DNA replication in cyanobacteria.

生物钟确保蓝细菌中 DNA 的成功复制。

• 发表时间: 2021-05-18
• 影响因子: 11.1
• 作者: Liao, Yi;Rust, Michael J
• 通讯作者: Rust, Michael J

Kalman-like Self-Tuned Sensitivity in Biophysical Sensing.

• DOI: 10.1016/j.cels.2019.08.008
• 发表时间: 2019-11-27
• 期刊: Cell systems
• 影响因子: 9.3
• 作者: Kabir B Husain;Weerapat Pittayakanchit;Gopal Pattanayak;Michael J Rust;A. Murugan
• 通讯作者: A. Murugan