Determining the mechanistic links between the metabolic and cell division cycles

描述(申请人提供)：细胞协调新陈代谢、生长和细胞分裂周期的调节，以响应环境。例如，为了应对不利的环境条件，酵母和其他生物会放慢生长速度，调节新陈代谢以储存碳水化合物，而碳水化合物对抗逆性和长期生存至关重要。酵母细胞周期性地利用和重建这些碳水化合物储备，这是被称为“代谢循环”的现象的一个关键因素。基于细胞分裂与代谢周期的特定阶段相关的观察，有人提出细胞周期受代谢周期的调控。然而，调控的层级和碳水化合物储存代谢对细胞周期控制的贡献仍然不清楚，并存在着激烈的争论。我们的目标的成功完成将导致解决代谢和细胞分裂周期是如何机械地联系在一起的，以及它们的调节是分级的还是相互依赖的。为了实现我们的目标，我们将利用微流体以及现代成像和信号处理方法来开发一个新的实验平台，用于研究单个酵母细胞的代谢循环。微流控培养技术的应用为新陈代谢研究提供了独特的优势。与现有方法不同，微流控培养过程中的快速和持续流动允许直接指定胞外营养浓度，而不依赖于细胞密度。因此，我们预计我们的创新低成本平台将被从事代谢调节工作的研究人员社区广泛采用。我们的具体目标包括：(AIM1)可视化单细胞的代谢和细胞分裂周期；b开发代谢调节的荧光报告程序，与现有的细胞周期报告程序一起使用；(AIM2)将这套报告程序与遗传和化学操作信号通路结合使用，以确定代谢和细胞周期调节之间的分子机制(S)。与公共健康相关：在人类中，尽管几十年前就描述了沃伯格效应--有氧糖酵解的上调--以及癌症中的其他代谢障碍，但其潜在的分子机制才逐渐被发现。具体地说，目前尚不清楚代谢变化是肿瘤发生的原因还是结果。解开酵母等更易处理的生物体中的代谢过程，可以极大地促进它们在复杂的人类系统中的理解，从而有助于识别影响新陈代谢的新药物靶点。 与公共健康相关：细胞协调新陈代谢、生长和细胞分裂周期的调节，以响应环境。我们的目标是确定代谢和细胞分裂周期是如何机械地联系在一起的，以及它们的调节是分级的还是相互依赖的。

DESCRIPTION (provided by applicant): Cells coordinate the regulation of metabolism, growth and the cell division cycle in response to the environment. For example, in response to unfavorable environmental conditions, yeast and other organisms slow down growth and regulate their metabolism to store carbohydrates, which are crucially important for stress resistance and long-term survival. Yeast cells periodically utilize and rebuild these carbohydrate reserves, which is a key element of the phenomenon known as "metabolic cycling". Based on the observation that cell division is correlated with specific phases of the metabolic cycle, it ha been proposed that the cell cycle is gated by the metabolic cycle. However, the hierarchy of regulation and the contribution of carbohydrate storage metabolism to cell cycle control remain unclear and heavily debated. Successful completion of our aims will result in resolving how the metabolic and the cell division cycle are mechanistically linked and whether their regulation is hierarchical or interdependent. To achieve our aims, we will employ microfluidics and modern imaging and signal processing methods to develop a novel experimental platform for examining metabolic cycling in individual yeast cells. The application of microfluidic cultivation offers a unique advantage for studying metabolism. In contrast to existing methods, rapid and constant flow during microfluidic cultivation allows direct specification of extracellular nutrient concentrations independent of cell density. Thus, we expect our innovative low-cost platform to be widely adopted by the community of researchers working on metabolic regulation. Our specific aims include: (Aim1) Visualize the metabolic and the cell division cycle in single cells b developing fluorescent reporters for metabolic regulation to be used together with existing reporters for the cell cycle; (Aim2) Use this set of reporters in conjunction with genetic and chemical manipulations of signaling pathways to determine the molecular mechanism(s) linking metabolic and cell cycle regulation. Relevance to public health: In humans, despite the fact that the Warburg Effect -the upregulation of aerobic glycolysis- and other metabolic malfunctions in cancer were described decades ago, the underlying molecular mechanisms are only gradually being uncovered. Specifically, it remains unclear whether metabolic alterations are the cause or an effect of oncogenesis. Unraveling metabolic processes in more tractable organisms, such as yeast, can greatly facilitate their understanding in complex human systems and thereby contribute to identifying novel drug targets affecting metabolism. PUBLIC HEALTH RELEVANCE: Cells coordinate the regulation of metabolism, growth and the cell division cycle in response to the environment. We aim to determine how the metabolic and the cell division cycle are mechanistically linked and whether their regulation is hierarchical or interdependent.