Quantitative real-time characterization of single-cell aging: from phenotypes to

描述（由申请人提供）：细胞衰老是细胞中遗传和分子变化积累的动态过程。与年龄相关的细胞结构损伤会导致生理功能的退化，从而导致细胞死亡。癌症、2型糖尿病和阿尔茨海默病等多种疾病都与细胞衰老有关；然而，我们对细胞衰老机制的理解以及这些机制如何与各种疾病状态的启动相结合是非常有限的。例如，我们对染色体不稳定是如何在老细胞中发生的知之甚少。对直接调节寿命的一组基因和基因网络以及在这种调节中使用的机制也缺乏基本的了解。细胞衰老是一种复杂的表型，需要采用新颖的实验方法和技术平台来进行全面的衰老研究。以酿酒酵母的复制老化为实验模型，提出采用定量单细胞和单分子跟踪方法，目的是：(1)揭示双稳态半乳糖代谢对单细胞老化的影响；(2)研究染色体如何随着衰老而变得不稳定；(3)探索单细胞中细胞老化与蛋白质错误折叠之间的联系。为了方便复制寿命的实时测量，我们将利用微流体平台，使子细胞与母细胞自动分离。衰老的母细胞将被时间动态成像，直到它们不再产生子细胞。这些项目的结果将通过阐明哪些遗传和表型变化伴随或驱动衰老过程，拓宽我们对单细胞如何衰老的有限理解。几十年来，传统微机械臂老化平台的劳动强度限制了老化领域的研究进展。基于菌落的衰老测定无法在单细胞水平上量化衰老表型是另一个重要的缺陷，因为通常存在细胞间的变异

DESCRIPTION (provided by applicant): Cellular aging is the dynamic process of accumulating genetic and molecular changes in cells. Age-associated damage to cellular structures results in the deterioration of physiological functions, leading to cell death. A variey of diseases such as cancer, type-2 diabetes, and Alzheimer's disease are linked to cellular aging; yet, our understanding into the mechanisms of cellular aging and how these mechanisms are coupled to the initiation of various disease states is very limited. For example, we know very little about how chromosome instabilities occur in old cells. A basic understanding on the set of genes and gene networks responsible from directly regulating lifespan and the mechanisms used in this regulation is also missing. This lack of understanding is contributed by the fact that cellular aging is a complex phenotype to measure and comprehensive studies on aging require the application of novel experimental approaches and technological platforms. Using the replicative aging of the yeast Saccharomyces cerevisiae as an experimental model, I propose to apply quantitative single-cell and single-molecule tracking approaches with the goal of: (1) uncovering the effect of bistable galactose metabolism on single-cell aging; (2) investigating how chromosomes become unstable with aging; (3) exploring the links between cellular aging and protein misfolding in single cells. To facilitate real-time measurements of replicative lifespan, we will utilize a microfluidics platform that automates the separation of daughter cells away from their mothers. Aging mother cells will be time-dynamically imaged until they no longer produce daughter cells. Results from these projects will broaden our limited understanding on how single-cells age by elucidating which genetic and phenotypic changes accompany or drive the aging process. For several decades, the labor-intensive nature of the conventional micromanipulator-based aging platforms has limited the research progress in the aging field. The inability of the colony-based aging assays to quantify aging phenotypes at the single-cell level was another important deficiency, as there are usually cell-to-cell variations in aging among the cells forming a colony. Using microfluidics platforms to automate single-cell lifespan measurements will overcome these deficiencies and limitations, and has the potential to transform the field of aging.