Determining the mechanistic links between the metabolic and cell division cycles

描述（申请人提供）：细胞根据环境协调调节代谢、生长和细胞分裂周期。例如，在不利的环境条件下，酵母和其他生物会减缓生长并调节新陈代谢以储存碳水化合物，这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”现象的关键因素。根据细胞分裂与代谢周期的特定阶段相关的观察，有人提出细胞周期是由代谢周期控制的。然而，调控的层次结构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，并且存在大量争论。成功完成我们的目标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层的还是相互依赖的。为了实现我们的目标，我们将采用微流体和现代成像和信号处理方法来开发一个新的实验平台，用于检查单个酵母细胞的代谢循环。微流体培养的应用为研究代谢提供了独特的优势。与现有方法相比，微流体培养过程中快速和恒定的流动允许独立于细胞密度的细胞外营养浓度的直接规范。因此，我们希望我们创新的低成本平台能够被从事代谢调节的研究人员广泛采用。我们的具体目标包括：（目的1）可视化单细胞的代谢和细胞分裂周期；b开发用于代谢调控的荧光报告基因，与现有的细胞周期报告基因一起使用；（目的2）将这组报告基因与信号通路的遗传和化学操作结合使用，以确定连接代谢和细胞周期调节的分子机制。与公众健康相关：在人类中，尽管沃伯格效应（有氧糖酵解的上调）和癌症中的其他代谢障碍在几十年前就被描述过，但其潜在的分子机制只是逐渐被揭示出来。具体来说，目前尚不清楚代谢改变是肿瘤发生的原因还是结果。在更容易处理的生物（如酵母）中揭示代谢过程，可以极大地促进它们在复杂人体系统中的理解，从而有助于确定影响代谢的新型药物靶点。

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.