The Biochemistry of Clock Function in Fluctuating Environments

波动环境中时钟功能的生物化学

基本信息

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
项目类别：
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

项目摘要

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，近24小时的时间是由内部生物钟。在许多生物体中，当昼夜节律时，健康和健身会受到损害时钟与外部环境中的每日周期没有适当同步。因此了解时钟如何应对挑战性波动环境的响应至关重要现代生活典型的不规则投入。这个问题在概念上具有挑战性，因为昼夜节律是复杂的系统。通常，有一个由生化的核心振荡器产生节奏每日信号的电路，并且可以通过输入来调整此节奏的时间将有关环境信息传达给振荡器的信息的信号。但是，这个振荡器还嵌入了细胞生理的其余部分中，因此其对环境不断变化的反应是可能取决于代谢和其他信号通路的状态。我们正在使用细菌模型有机体元素弹药破解了时钟 - 环境相互作用的问题因为该生物具有一个显着的特征，即可以在体外重构核心振荡器使用纯化的蛋白质。因此，我们将使用简化的方法来建立综合的当受到环境波动的影响时，完整单元格中时钟功能的数学模型。在 AIM 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在各种温度，代谢物浓度和蛋白质上的核心时钟蛋白上石化的图表。使用高级统计方法，我们将约束基本模型发现温度补偿，代谢感应和夹带功能的反应核心振荡器。在AIM 2中，我们将研究时钟如何随着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隔离时钟灵敏度的历史依赖性核心振荡器不存在。然后，我们将使用遗传分析找到关键途径用于在体内调节时钟灵敏度。然后将这些数据纳入扩展在环境条件下描述时钟在体内功能的数学模型波动。在AIM 3中，我们将开发一种深诱变扫描方法，以找到时钟表型同时，时钟基因中10,000点突变的竞争增长缺陷。这我们不仅会允许我们发现时钟蛋白上的关键交互位点，而且还可以获得一个破坏温度补偿的周期突变体和突变体的全面列表。因为该测定基于昼夜节律中无处不在的转录反馈循环，它可以是通常也应用于其他时钟系统。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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：
通讯作者：

The circadian clock ensures successful DNA replication in cyanobacteria.

DOI：
10.1073/pnas.2022516118
发表时间：
2021-05-18
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Liao Y;Rust MJ
通讯作者：
Rust MJ