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: 8297341
• 负责人: Natalie A Cookson
• 金额: $ 25.86万
• 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/8297341
• 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. PUBLIC HEALTH RELEVANCE: The aging process is governed by both the environment and genetics. Environments that provide sub-optimal levels of nutrients have been shown to greatly increase life span in numerous model organisms, from yeasts to mammals. We will develop a highly parallel microfluidic platform to observe and characterize the interplay between environmental and genetic factors that affect important aspects of eukaryotic aging.

描述（由申请人提供）：衰老是一个受遗传和环境因素共同影响的复杂过程，“变老”的负面影响可以表现为多种形式。单个细胞（例如神经元和干细胞）的寿命影响多细胞生物衰老的速度和优雅程度。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。然而，尽管许多与衰老相关的重要途径已被确定，但限制寿命的基本机制仍不清楚。这项研究的一个障碍是追踪长期行为的困难，不仅在人类和其他长寿哺乳动物中，而且在更简单的模式生物中也是如此。虽然单细胞酿酒酵母是最简单的衰老模型，并且最适合遗传和分子操作，但即使在这种快速生长的生物体中，现有的监测衰老的方法仍然有限。 我们建议使用微流控技术作为研究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有很大影响，我们将开发一种高度并行的微流体装置，能够将不同的细胞群置于动态环境中。我们将其与新的图像处理技术相结合，从而能够观察在静态和动态环境中生长的单细胞的老化动态。该平台的优势在于，可以生成个体生物体从出生到老年的终生统计数据。我们将展示该平台的潜力，通过关注衰老的关键决定因素——热量摄入，为长期动态提供新的见解。我们将首先使用微流体梯度平台将大量细胞置于一系列静态葡萄糖浓度下，描述静态热量限制 (CR) 对寿命的影响。由于 CR 可能不需要恒定即可延长寿命，因此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的反应机制。 遗传因素对衰老也有很大影响。一生中基因突变的积累会导致癌症等与衰老相关的疾病的发生。酵母是这种现象的一个令人惊讶的有用模型，因为观察到母细胞在达到临界细胞分裂次数时会切换到基因组不稳定状态。这种转换导致杂合性丢失（LOH）事件频繁发生。我们将开发一种使用双色荧光显微镜来跟踪 LOH 事件的方法，并且我们将使用我们的微流体平台来观察响应 CR 的 LOH 频率的变化。最后，酵母的代谢循环（以氧化还原状态的振荡为代表）已被证明受到与寿命延长有关的途径的调节。我们将使用一种修饰的荧光蛋白来感知氧化状态以及我们的动态微流体平台，以确定寿命与酵母代谢振荡周期的关系。 公共健康相关性：衰老过程受环境和遗传因素共同影响。事实证明，提供次优营养水平的环境可以大大延长从酵母到哺乳动物等多种模式生物的寿命。我们将开发一个高度并行的微流体平台来观察和表征影响真核衰老重要方面的环境和遗传因素之间的相互作用。