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

项目摘要：新陈代谢节律发生在生物体内不同的细胞和隔间。这个 这些节律的起源和对其他生物振荡器(如细胞周期、生物钟)的影响只是 开始被理解了。申请者的长期目标是了解机制、功能和 真核生物模式芽生酵母代谢节律与细胞周期的相互作用。酵母菌的代谢 周期(YMC)是一种发生在恒化器中的同步代谢节律。人口同步性出现了 通过分泌的代谢产物在细胞之间进行YMC到YMC的偶联。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事件。这两个 振荡器有不同的周期，但仍然保持协调，以至于一部分人致力于 疾病预防控制中心每一位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 事件通常是在不同类型的人群中进行测量和平均的，这掩盖了 发生在单个细胞中。这项提议的目的是获得对这些细胞内振荡器的新见解。 通过测量和扰动单个细胞的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加强 反馈循环(即，进入碳分解代谢状态会触发细胞周期，并反过来启动细胞 循环触发进入碳分解代谢状态)。申请者将生成寻址中心的数据 假说及其替代方案有三个具体目的：(1)建立荧光报告分析方法来测量 从循环恒化器获取的单个细胞代谢和细胞周期状态的群体快照；(2) 扰乱强化反馈回路以扰乱自行车中YMC和CDC事件的同步 恒化器；以及(3)利用时间推移测量和扰动恒化器外的YMC-CDC动态 微流控荧光显微镜。目标1将阐明代谢和代谢的时间和协调 循环恒化器中不同生长条件下的细胞周期事件。Aim 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 在没有通过分泌的代谢物进行细胞间交流的情况下，代谢节律发生的程度 以及它们是否像在恒化器中看到的那样与细胞周期事件保持协调。观察到的是 碳分解代谢和细胞周期进入在不同的生长条件下保持协调 会强烈支持中心假说。这项工作具有创新性，因为它结合了单细胞 解决酵母中与代谢广泛相关的悬而未决问题的技术和分子遗传学 其他真核生物的节律和细胞周期。这一提议意义重大，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和保持的调控原则 功能齐全。

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.