Molecular and cellular mechanisms of circadian timekeeping in a prokaryote model

 描述(申请人提供)：这个项目解决了两个基本问题：生物钟如何在分子水平上作为计时机制发挥作用，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？生物钟是一个振荡计时器，它驱动从细菌到人类的不同生物体的24小时生物活动节奏，其基本分子机制的中断对健康产生不利影响。人类的生物钟功能障碍与一系列健康状况有关，如心血管疾病、癌症、代谢综合征、精神疾病和睡眠障碍。尽管在蓝藻和哺乳动物之间进化出了不同的计时策略，但蓝藻聚球藻的生物钟产生了真正的遗传、生理和代谢活动的昼夜节律，满足了定义真核生物生物钟的所有标准。对苏云金杆菌的生物钟有一个定量的、系统水平的、生化的理解。 它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重组。在这种遗传上易于处理的模式生物中，有可能系统地改变时钟蛋白的物理和生化特性，并通过蛋白质相互作用网络追踪这些变化的影响，从它们的近端效应到表达的昼夜节律表型。此外，正如在哺乳动物细胞中一样，长链球菌的生物钟控制着细胞分裂的时间。该项目将利用最近的概念和技术进步，以前所未有的清晰度来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进行折叠，并成为振荡器和输出通路之间的连接器，以及振荡器蛋白质对代谢物进行采样以设置当地时间的发现，这些发现将使建立更完整的体外时钟成为可能，该时钟显示出与基因表达控制相关的有节奏的输出。利用这种准备，以及来自BioLayer干涉测量的合作伙伴互动的动力学测量，该项目将量化有助于计时、同步和有节奏输出的步骤。在体内分析改变这种相互作用的突变将把特定的步骤与时钟功能联系在一起。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。在精通或缺乏对细胞分裂的时钟控制的遗传背景中，对携带荧光标记的时钟蛋白和昼夜节律标记的分裂细胞进行延时测量，将提供对时钟成分如何与正确的时间戳遗传的洞察。蛋白质组学方法将确定负责细胞内时钟成分定位的合作伙伴，以及时钟允许或取消胞质分裂的能力。总之，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。

 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.