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（非- oid -09-058，通知标题：NIH宣布为竞争性修订申请提供恢复法案基金），资助1 R01 GM076536-01，“用于单细胞全基因组蛋白质和RNA定位的细胞芯片”。蛋白质可能组织成复合体，根据细胞的需要进行组装和拆卸。然而，蛋白质也必须避免灾难性聚集，这是许多重要疾病（如阿尔茨海默氏症和朊病毒疾病）甚至衰老本身的基础。这一竞争性修订遵循了我们最近在研究这项资助的主要目标时的一个惊人发现：数百种不同的、正常的细胞质蛋白物种在酵母酵母静止细胞中形成代谢物诱导的、可逆的、宏观的点状灶。这些宏观组合是功能性的还是灾难性的？这个问题的答案对于我们理解静止细胞和聚集性疾病是很重要的。如果这些组合是功能性的或含有功能性蛋白，这代表了静止细胞中意想不到的高水平组织，这对大多数处于静止状态的人类细胞具有重要影响。如果这些组合代表广泛的聚集，这些结构可能是聚集相关疾病和衰老的重要因素。在本次修订中，我们提出了区分这两种假设的实验，目的一是测量点状灶形成和溶解的动力学，将动力学特性与蛋白质的功能和序列特性联系起来，目的二是合理设计和表达具有改变聚集倾向的蛋白质，并监测点状灶的形成。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，并监测点状灶的形成，进一步评估聚集假说。最后，目的3是应用显微镜确定哪些蛋白质在静止细胞中共定位到相同的点状病灶，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具体的证据，在蛋白质-蛋白质的基础上，在静止细胞中形成功能性蛋白质组装或灾难性聚集，因此将加快我们原始资助的研究节奏，以及招募/留住符合恢复法案目标的科学家。

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.