Cell chips for genome-wide protein and RNA localization in single cells

描述(由申请人提供)：这是一个竞争性修订(NOT-OD-09-058，通知标题：NIH宣布为竞争性修订申请提供恢复法案资金)，授予1 R01 GM076536-01，“用于单个细胞中全基因组蛋白质和RNA定位的细胞芯片”。蛋白质可能会组织成复合体，根据细胞的需要进行组装和拆解。然而，蛋白质也必须避免灾难性的聚集，这是许多重要疾病(例如阿尔茨海默氏症和普里恩病)甚至衰老本身的基础。这一竞争性修订是我们最近在研究这项赠款的主要目标期间取得的一个惊人发现：数百种不同的、通常是胞质的蛋白质物种在酵母S.cerevisae的静止细胞中形成代谢物诱导的、可逆的宏观点状焦点。这些宏观组装是正常的还是灾难性的？这个问题的答案对于我们理解静止细胞和基于聚集的疾病很重要。如果这些组装是有功能的或含有功能蛋白质，这代表了静止细胞中出人意料的高水平组织，对主要处于静止状态的人类细胞具有重要的影响。如果这些集合体代表广泛的聚集，那么这种结构很可能是聚集相关疾病和衰老的重要因素。在这次修订中，我们提出了区分这两个假设的实验，目的1测量点状焦点的形成和溶解的动力学，将动力学性质与蛋白质的功能和序列性质联系起来，目的2合理设计和表达具有改变聚集倾向的蛋白质，并监测点状焦点的形成。我们的目标是通过计算设计具有非天然聚集倾向的蛋白质，在酿酒酵母中表达这些蛋白质，并监测点状焦点的形成，从而进一步评估聚集假说。最后，目标3是应用显微镜来确定哪些蛋白质共定位于静止细胞中的相同点状焦点，从而测试来自相同途径的蛋白质是否共定位，支持功能组装。这些目标的结果将是在蛋白质基础上的具体证据，无论是在静止细胞中形成功能性蛋白质集合，还是灾难性的聚集，因此将加快我们最初拨款的研究速度，以及招募/留住符合复苏法案目标的科学家。 与公共卫生相关：蛋白质必须组织成复合体，根据细胞需要进行组装和拆解，同时避免灾难性的聚集。这种聚集是许多重要疾病的基础，包括阿尔茨海默氏症和普里恩病，甚至衰老本身。这项拨款建议进行细胞生物学和成像实验，以了解在静止细胞中观察到的广泛、可逆的蛋白质聚集的机制基础。这项工作将增加我们对蛋白质聚集的机制和环境的理解，从而朝着更好地理解聚集性疾病和衰老背后的力量迈出了一步。

DESCRIPTION (provided by applicant): This is a competing revision (NOT-OD-09-058, Notice title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications) for grant 1 R01 GM076536-01, "Cell chips for genome-wide protein and RNA localization in single cells". Proteins are likely to organize into complexes that assemble and disassemble depending on cellular needs. However, proteins must also avoid catastrophic aggregation, which underlies many important diseases (e.g., Alzheimer's & prion diseases) and even aging itself. This competitive revision pursues a striking discovery we recently made during work on the primary aims of this grant: hundreds of distinct, normally cytosolic protein species form metabolite-induced, reversible, macroscopic punctate foci in quiescent cells of the yeast S. cerevisae. Are these macroscopic assemblies functional or catastrophic? The answers to this question are important for our understanding of quiescent cells and of aggregation-based disease. If these assemblies are functional or contain functional proteins, this represents unexpectedly high levels of organization in quiescent cells, with important ramifications for human cells, which are mostly in the quiescent state. If these assemblies represent widespread aggregation, such structures are likely important factors for aggregation-related disease and aging. In this revision, we propose experiments to distinguish these two hypotheses, with Aim 1 to measure kinetics of formation and dissolution of the punctate foci, relating kinetic properties to functional and sequence properties of the proteins, Aim 2 to rationally design & express proteins with altered aggregation propensities in yeast and monitor the formation of punctate foci. Our goal is to further assess the aggregation hypothesis by computationally designing proteins with a non-native aggregation propensity, expressing these proteins in S. cerevisiae, and monitoring formation of punctate foci. Finally, Aim 3 is to apply microscopy to determine which proteins co-localize to the same punctate foci in quiescent cells, thus testing if proteins from the same pathways co-localize, supporting functional assemblies. The results of these Aims will be concrete evidence, on a proteinby- protein basis, either for forming functional protein assemblies in quiescent cells or for catastrophic aggregation, and will thus accelerate the tempo of research in our original grant, as well as recruiting/retaining scientists consistent with Recovery Act aims. PUBLIC HEALTH RELEVANCE: Proteins must organize into complexes that assemble and disassemble depending on cellular needs, while simultaneously avoiding catastrophic aggregation. Such aggregation underlies many important diseases, including Alzheimer's disease and prion diseases, and even aging itself. This grant proposes cell biology and imaging experiments to understand the mechanistic basis for widespread, reversible protein aggregation observed in quiescent cells. This work will increase our understanding of the mechanisms and circumstances under which proteins aggregate, and thus will be a step towards better understanding of the forces underlying aggregation diseases and aging.