Measuring and perturbing metabolic rhythms and the cell division cycle in single cells

测量和扰乱单细胞的代谢节律和细胞分裂周期

• 批准号: 9901540
• 负责人: NICOLAS EMILE BUCHLER
• 金额: $ 29.68万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9901540
• 关键词: Acetyl Coenzyme A; Address; Alleles; Biochemical; Biological Assay; Biological Pacemakers; Carbon; Catabolism; Cell Communication; Cell Compartmentation; Cell Cycle; Cells; Circadian Rhythms; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Disease Progression; Enzymes; Eukaryota; Event; Feedback; Fluorescence Microscopy; Frequencies; Genetic; Glycogen Phosphorylase; Goals; Growth; Immobilization; Individual; Lead; Link; Masks; Measures; Metabolic; Microfluidics; Mitosis; Modeling; Molecular Genetics; Mutation; NADH; Organism; Oxidation-Reduction; Pathway interactions; Penetrance; Phase; Population; Population Heterogeneity; Public Health; Regulation; Reporter; Research; Saccharomycetales; Series; Signal Pathway; Testing; Trehalase; Trehalose; Work; Yeasts; circadian; circadian pacemaker; cyclin G1; event cycle; experimental study; human disease; innovation; insight; mathematical model; novel therapeutic intervention; peroxiredoxin; public health relevance; single cell technology

PROJECT ABSTRACT: Metabolic rhythms occur in different cells and compartments within organisms. The origins and impact of these rhythms on other biological oscillators (e.g. cell cycle, circadian clocks) is only starting to be understood. The applicant’s long-term goal is to understand the mechanisms, function, and interaction of metabolic rhythms and the cell cycle in budding yeast, a model eukaryote. The yeast metabolic cycle (YMC) is a synchronous metabolic rhythm that occurs in a chemostat. The population synchrony arises from YMC-to-YMC coupling between cells via secreted metabolites. The YMC within each cell also interacts with the cell division cycle (CDC) to coordinate the events of carbon catabolism and cell cycle entry. These two oscillators have different periods, yet remain coordinated such that a fraction of the population commits to the CDC each YMC. Inference of YMC-CDC dynamics has been challenging because metabolic and cell cycle events are often measured and averaged across a heterogeneous population, which masks the dynamics that occur in a single cell. The objective of this proposal is to obtain new insights into these intracellular oscillators by measuring and perturbing the YMC and CDC in single cells. The central hypothesis is that the YMC and CDC can oscillate independently of one another but are normally coordinated in yeast through a reinforcing feedback loop (i.e., entry into a carbon catabolic state triggers the cell cycle, and, reciprocally, initiating the cell cycle triggers entry into a carbon catabolic state). The applicants will generate data that address the central hypothesis and its alternative with three specific aims: (1) Develop fluorescent reporter assays to measure population snapshots of metabolic and cell cycle states in single cells taken from a cycling chemostat; (2) Perturb the reinforcing feedback loop to disrupt the synchronization of YMC and CDC events in a cycling chemostat; and (3) Measure and perturb YMC-CDC dynamics outside the chemostat using timelapse fluorescence microscopy with microfluidics. Aim 1 will elucidate the timing and coordination of metabolic and cell cycle events in a cycling chemostat across different growth conditions. Aim 2 will directly test the reinforcing feedback loop that coordinates these oscillators in a cycling chemostat. Aim 3 will measure the extent to which metabolic rhythms occur in the absence of cell-to-cell communication via secreted metabolites and whether they remain coordinated with cell cycle events as seen in the chemostat. The observation that carbon catabolism and cell cycle entry remain coordinated in single cells across diverse growth conditions would strongly support the central hypotheses. This work is innovative because it combines single-cell technology and molecular genetics to address an unsolved problem in yeast with broad relevance to metabolic rhythms and cell cycle in other eukaryotes. This proposal is significant because it elucidates new mechanisms and regulatory principles of how intracellular oscillators with different frequencies can interact and remain functional.

项目摘要：代谢节律发生在生物体的不同细胞和区室中。这 这些节律的起源和对其他生物振荡器（例如细胞周期、生物钟）的影响仅是 开始被理解。申请人的长期目标是了解机制、功能和 芽殖酵母（一种模型真核生物）代谢节律与细胞周期的相互作用。酵母代谢 循环（YMC）是在恒化器中发生的同步代谢节律。人口同步出现 细胞之间通过分泌的代谢物进行 YMC-to-YMC 偶联。每个细胞内的 YMC 也相互作用 与细胞分裂周期（CDC）协调碳分解代谢和细胞周期进入的事件。这两个 振荡器有不同的周期，但保持协调，使得一小部分人口致力于 CDC各YMC。 YMC-CDC 动力学的推断一直具有挑战性，因为代谢和细胞周期 事件通常是在异质人群中进行测量和平均，这掩盖了事件的动态 发生在单个细胞中。该提案的目的是获得对这些细胞内振荡器的新见解 通过测量和扰动单细胞中的 YMC 和 CDC。中心假设是 YMC 和 CDC 可以相互独立地振荡，但通常在酵母中通过一种增强因子进行协调 反馈循环（即进入碳分解代谢状态会触发细胞周期，并且反过来，启动细胞 循环触发进入碳分解代谢状态）。申请人将生成解决中央问题的数据 假设及其替代方案具有三个具体目标：（1）开发荧光报告测定法来测量 从循环恒化器获取的单细胞代谢和细胞周期状态的群体快照； (2) 扰乱强化反馈循环，破坏自行车中 YMC 和 CDC 事件的同步 恒化器； (3) 使用延时测量和扰乱恒化器外的 YMC-CDC 动力学 具有微流体技术的荧光显微镜。目标 1 将阐明代谢和代谢的时间安排和协调 不同生长条件下循环恒化器中的细胞周期事件。目标 2 将直接测试 加强反馈回路，协调循环恒化器中的这些振荡器。目标 3 将测量 在没有通过分泌代谢物进行细胞间通讯的情况下代谢节律发生的程度 以及它们是否与恒化器中看到的细胞周期事件保持协调。观察结果表明 在不同的生长条件下，单细胞中的碳分解代谢和细胞周期进入保持协调 强烈支持中心假设。这项工作具有创新性，因为它结合了单细胞 技术和分子遗传学来解决酵母中与代谢广泛相关的未解决问题 其他真核生物的节律和细胞周期。该提案意义重大，因为它阐明了新机制 不同频率的细胞内振荡器如何相互作用和保持的调节原理 功能性的。