Determining the mechanistic links between the metabolic and cell division cycles

描述（由申请人提供）：细胞协调代谢、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，酵母和其他生物减缓生长并调节其代谢以储存碳水化合物，这对抗应激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”现象的关键要素。基于细胞分裂与代谢周期的特定阶段相关的观察，已经提出细胞周期由代谢周期门控。然而，调节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，并存在很大争议。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，以及它们的调节是否是分层的或相互依赖的。为了实现我们的目标，我们将采用微流体技术和现代成像和信号处理方法来开发一种新的实验平台，用于检查单个酵母细胞中的代谢循环。微流控培养技术的应用为代谢研究提供了独特的优势。与现有方法相比，微流体培养期间的快速和恒定流动允许直接指定细胞外营养物浓度，而不依赖于细胞密度。因此，我们希望我们的创新低成本平台能够被代谢调节研究人员广泛采用。我们的具体目标包括：（Aim 1）通过开发用于代谢调节的荧光报告物与用于细胞周期的现有报告物一起使用，可视化单细胞中的代谢和细胞分裂周期B;（Aim 2）将这组报告物与信号传导途径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。与公共卫生的相关性：在人类中，尽管瓦尔堡效应--有氧糖酵解的上调--和癌症中的其他代谢功能障碍在几十年前就被描述过，但潜在的分子机制只是逐渐被揭示出来。具体而言，代谢改变是肿瘤发生的原因还是影响仍不清楚。在更易处理的生物体（如酵母）中解开代谢过程可以极大地促进对复杂人类系统的理解，从而有助于确定影响代谢的新药物靶点。

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.