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

项目摘要：代谢节律发生在生物体的不同细胞和区室中。的 这些节律的起源和对其他生物振荡器（例如细胞周期，昼夜节律钟）的影响只是 开始被理解。申请人的长期目标是了解机制，功能， 芽殖酵母（一种模式真核生物）中代谢节律和细胞周期的相互作用。酵母代谢 周期（YMC）是发生在恒化器中的同步代谢节律。人口同步性出现 通过分泌的代谢物在细胞之间进行YMC-至-YMC偶联。每个细胞内的YMC也相互作用 与细胞分裂周期（CDC）协调碳催化剂和细胞周期进入事件。这两 振荡器具有不同的周期，但仍保持协调，使得一小部分人口致力于 CDC每个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 事件通常是在异质群体中测量和平均的，这掩盖了 发生在单个细胞中。这个建议的目的是获得这些细胞内振荡器的新见解 通过测量和干扰单细胞中的YMC和CDC。核心假设是，YMC和 CDC可以相互独立地振荡，但通常在酵母中通过增强的 反馈回路（即，进入碳分解代谢状态触发细胞周期，并且，首先，启动细胞 循环触发进入碳分解代谢状态）。申请人将生成数据， 假设及其备选方案，具有三个具体目标：（1）开发荧光报告基因测定， 从循环恒化器获取的单细胞中的代谢和细胞周期状态的群体快照;（2） 扰乱强化反馈回路，破坏循环中YMC和CDC事件的同步 恒化器;和（3）使用时间流逝测量和扰动恒化器外部的YMC-CDC动力学 荧光显微镜和微流体技术。目的1将阐明代谢和代谢的时间和协调， 在不同的生长条件下，在循环恒化器中的细胞周期事件。目标2将直接测试 在循环恒化器中协调这些振荡器的加强反馈回路。目标3将衡量 在缺乏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所见的细胞周期事件保持协调。的观察结果 在不同的生长条件下，单细胞中碳催化剂和细胞周期进入保持协调 会强烈支持中心假设。这项工作是创新的，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的问题 其他真核生物的细胞周期和节律。这一建议意义重大，因为它阐明了新的机制 以及不同频率的细胞内振荡器如何相互作用并保持 不降低

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.