Molecular and cellular mechanisms of circadian timekeeping in a prokaryote model

原核生物模型中昼夜节律的分子和细胞机制

基本信息

批准号：
9253415
负责人：
SUSAN S GOLDEN
金额：
$ 59.42万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-04 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9253415
关键词：
Address Affect Animal Model Animals Bacteria Biochemical Biological Clocks Biological Models Biological Rhythm Cardiovascular Diseases Cell Cycle Cell division Cells Circadian Rhythms Clock protein Cues Cyanobacterium Cytokinesis Disease Environment Eukaryota Exhibits Functional disorder Gene Expression Genetic Health Human In Vitro Inherited Interferometry Kinetics Label Malignant Neoplasms Mammalian Cell Mammals Measurement Mental disorders Metabolic Metabolic syndrome Modeling Molecular Mutation Organism Output Pathway interactions Periodicity Phenotype Physiological Preparation Prokaryotic Cells Property Protein Biochemistry Proteins Proteomics Sampling Sleep Disorders Synechococcus System Time circadian pacemaker fitness in vivo insight pathogen protein biomarkers public health relevance reconstitution transcription factor

项目摘要

DESCRIPTION (provided by applicant): This project addresses two fundamental questions: how does a circadian clock function at the molecular level as a timekeeping mechanism, and how is it integrated at the cellular level to control activities such as gene expression and cell division? The circadian clock is an oscillatory timer that drives 24-h rhythms of biological activities in diverse organisms from bacteria to humans, and disruptions in its underlying molecular mechanism adversely affect fitness. Clock dysfunction in humans is related to a spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and sleep disorders. Despite different strategies for timekeeping that have evolved between cyanobacteria and mammals, the circadian clock of the cyanobacterium Synechococcus elongatus generates bona fide circadian rhythms of genetic, physiological, and metabolic activities that fulfill all criteria that define circadian clocks in eukaryotes. A quantitative, systems- level, biochemical understanding is attainable for the circadian clock of S. elongatus, whose fundamental circadian oscillator can be reconstituted in vitro with three proteins, KaiA, KaiB, and KaiC. In this genetically tractable model organism it is possible to systematically alter the physical and biochemical properties of clock proteins and trace the impact of these changes from their proximal effects, through the protein-interaction network, to the expressed circadian phenotype. Moreover, as is true in mammalian cells, the circadian clock of S. elongatus controls the timing of cell division. This project will leverage recent conceptual and technical advances to determine the mechanism of the timekeeping system with unprecedented clarity, understand how the clock controls activities in the cell, and elucidate how a sense of time is inherited when cells divide. The discoveries that KaiB refolds as part of the timekeeping mechanism and becomes a connector between oscillator and output pathways, and that metabolites are sampled by oscillator proteins to set the clock with local time, will enable establishment of a more complete in vitro clock that exhibits rhythmic output relevant for control of gene expression. Using this preparation, and kinetics measurements of partner interactions from BioLayer Interferometry, the project will quantify the steps that contribute to timekeeping, synchronization, and rhythmic output. Analysis in vivo of mutations that alter such interactions will tie specific steps to clock functions. The biochemical basis of interactions between two transcription factors that integrate temporal and environmental cues will be clarified. Time-lapse measurements of dividing cells that carry fluorescently labeled clock proteins and markers of the circadian cycle, in genetic backgrounds that are proficient or deficient in clock-control of cell division, will provide insight into how clock components are inherited with the correct timestamps. Proteomic approaches will identify partners responsible for localization of clock components within the cell and the ability of the clock to allow or disalow cytokinesis. Together, these approaches will elucidate clock mechanisms and the relationship between the circadian and cell division cycles.

描述（由申请人提供）：该项目解决了两个基本问题：生物钟如何在分子水平上发挥计时机制的作用，以及它如何在细胞水平上整合以控制基因表达和细胞分裂等活动？生物钟是一种振荡计时器，驱动从细菌到人类等多种生物体的生物活动的 24 小时节律，其潜在分子机制的破坏会对人类的健康产生不利影响，这与一系列因素有关。尽管蓝细菌和哺乳动物之间进化出了不同的计时策略，但蓝细菌细长聚球藻的生物钟产生了真正的遗传、生理和睡眠节律。满足定义真核生物生物钟的所有标准的代谢活动可以实现定量的、系统水平的、生化的理解。 S 的生物钟 elongatus 的基本昼夜节律振荡器可以在体外用三种蛋白质 KaiA、KaiB 和 KaiC 重建。在这种遗传上易于处理的模型生物中，可以系统地改变时钟蛋白质的物理和生化特性，并追踪这些变化的影响。它们通过蛋白质相互作用网络对表达的昼夜节律表型产生近端影响。此外，正如在哺乳动物细胞中一样，S. elongatus 的昼夜节律时钟也受到控制。该项目将利用最新的概念和技术进步，以前所未有的清晰度确定计时系统的机制，了解时钟如何控制细胞中的活动，并阐明细胞分裂时时间感是如何遗传的。 KaiB 作为计时机制的一部分重新折叠并成为振荡器和输出路径之间的连接器，以及振荡器蛋白对代谢物进行采样以根据本地时间设置时钟的发现，将使建立一个更完整的体外时钟成为可能。展示与基因表达控制相关的节律输出，并利用 BioLayer 干涉测量法对伙伴相互作用进行动力学测量，该项目将量化有助于计时、同步和改变节律输出的体内突变的步骤。将阐明整合时间和环境线索的两个转录因子之间相互作用的生化基础，这些细胞携带荧光标记的时钟蛋白和昼夜节律周期标记。在精通或缺乏细胞分裂时钟控制的遗传背景中，将深入了解时钟组件如何通过正确的时间戳遗传，并将识别负责细胞内时钟组件定位的合作伙伴以及时钟的能力。允许或禁止胞质分裂，这些方法将阐明时钟机制以及昼夜节律和细胞分裂周期之间的关系。