Aging of S. cerevisiae in a Dynamically Changing Environment

动态变化环境中酿酒酵母的老化

• 批准号: 8449265
• 负责人: Natalie A Cookson
• 金额: $ 27.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8449265
• 关键词: Address; Affect; Age; Aging; Aging-Related Process; Animal Model; Behavior; Biological Factors; Birth; Caloric Restriction; Cell division; Cells; Cellular Stress Response; Characteristics; Color; Complex; Computer software; Data; Development; Disease; Energy Intake; Environment; Environmental Risk Factor; Eukaryota; Event; Fluorescence; Fluorescence Microscopy; Frequencies; Gene Expression; Gene Mutation; Generations; Genetic; Genomic Instability; Glucose; Growth; Human; Image; Individual; Lead; Life; Longevity; Loss of Heterozygosity; Malignant Neoplasms; Mammals; Measurement; Measures; Mediating; Metabolic; Methods; Metric; Microfluidic Microchips; Microfluidics; Modeling; Molecular; Molecular Genetics; Monitor; Morphology; Mothers; Mutation; Neurons; Nutrient; Nutritional; Onset of illness; Organism; Oxidation-Reduction; Pathway interactions; Population; Process; Proteins; Research; Saccharomyces cerevisiae; Stem cells; Stress; Techniques; Technology; Time; Tweens; Yeasts; age related; image processing; imaging Segmentation; insight; response; statistics; tool; web site

DESCRIPTION (provided by applicant): Aging is a complex process governed by both genetic and environmental factors, and the negative effects of "growing old" can take on many forms. The life spans of individual cells, such as neurons and stem cells, influence the rate and grace with which multi-cellular organisms age. Nutritional stress and genetic instability have been identified as key determinants of life span in eukaryotes. However, while many important pathways involved in aging have been identified, the fundamental mechanisms that limit life span remain undefined. One hindrance to this research is the difficulty in tracking long-term behaviors, not just in humans and other long-lived mammals, but in simpler model organisms as well. While the single-celled S. cerevisiae is the least complicated model for aging and the most amenable to genetic and molecular manipulations, the existing methods for monitoring aging, even in this rapidly growing organism, remain limited. We propose to use microfluidic technology as an experimental platform for the study of aging in S. cerevisiae. As the growth environment has a large impact on the life span of eukaryotes, we will develop a highly parallel microfluidic device with the ability to subject separate populations of cells to a dynamic environment. We will combine this with new image processing techniques, enabling the observation of aging dynamics in single cells growing in both static and dynamic environments. This platform will have the advantage of generating life-long statistics for individual organisms as they progress from birth to old age. We will demonstrate the potential of this platform to provide new insight into long-term dynamics by focusing on a key determinant of aging, caloric intake. We will first characterize the effect of static Calorie Restriction (CR) on life span usinga microfluidic gradient platform to subject large populations of cells to a range of static glucose concentrations. Because CR may not need to be constant in order to extend life span, we will next investigate the effect of dynamic CR on longevity, in order to gain insight into the mechanisms by which an organism responds to low nutrient levels. Genetic factors also have a strong influence on aging. The accumulation of genetic mutations over the course of a lifetime leads to the onset of aging-related diseases, such as cancer. Yeast is a surprisingly useful model for this phenomenon, as mother cells are observed to switch to a state of genomic instability when they reach a critical number of cell divisions. This switch leads to the frequent occurrence of loss-of-heterozygosity (LOH) events. We will develop a method for employing two-color fluorescence microscopy to track LOH events, and we will use our microfluidic platform to observe changes in LOH frequency in response to CR. Finally, a metabolic cycle in yeast, manifested by oscillations in redox state, has been shown to be regulated by pathways involved in life span extension. We will use a modified fluorescent protein that senses oxidative state along with our dynamic microfluidic platform to determine how life span is related to the period of metabolic oscillations in yeast.

描述（由申请人提供）：衰老是一个由遗传和环境因素控制的复杂过程，“生长旧”的负面影响可以采用多种形式。单个细胞的生命跨度，例如神经元和干细胞，会影响多细胞生物年龄的速度和恩典。营养应激和遗传不稳定性已被确定为真核生物寿命的关键决定因素。但是，尽管已经确定了涉及衰老的许多重要途径，但限制寿命的基本机制仍然不确定。这项研究的一种障碍是难以跟踪长期行为，不仅是人类和其他长寿哺乳动物，而且在更简单的模型生物中。酿酒酵母单细胞链球菌是衰老的最不复杂的模型，也是最适合遗传和分子操作的模型，但即使在这种快速增长的生物体中，现有的监测衰老的方法仍然有限。 我们建议将微流体技术用作酿酒酵母衰老的实验平台。由于生长环境对真核生物的寿命具有很大的影响，因此我们将开发一种高度平行的微流体设备，能够将细胞种群分开到动态环境中。我们将与新的图像处理技术结合使用，从而使在静态和动态环境中生长的单个细胞中的老化动力学观察。该平台将具有为单个生物从出生到老年而产生终身统计数据的优势。我们将通过关注衰老，热量摄入的关键决定因素来展示该平台的潜力，以提供对长期动态的新见解。我们将首先将静态卡路里限制（CR）的影响对使用微流体梯度平台的寿命影响，从而使大量细胞群体成为一系列静态葡萄糖浓度。因为CR可能不需要持续就可以延长寿命，所以我们接下来将研究动态CR对寿命的影响，以便深入了解生物体对低营养水平反应的机制。 遗传因素对衰老也有很大影响。一生中遗传突变的积累导致与衰老有关的疾病（例如癌症）的发作。对于这种现象，酵母是一个令人惊讶的有用模型，因为当母细胞达到临界数量的细胞分裂时，观察到母细胞转变为基因组不稳定性的状态。该开关导致杂合性丧失（LOH）事件的频繁发生。我们将开发一种使用两色荧光显微镜跟踪LOH事件的方法，我们将使用微流体平台观察响应CR的LOH频率的变化。最后，已显示出氧化还原状态的振荡表现的酵母中的代谢周期已显示受生命跨度延长的途径的调节。我们将使用一种改良的荧光蛋白，该荧光蛋白与我们的动态微流体平台一起感知氧化态，以确定寿命与酵母中代谢振荡的时期如何相关。