High-throughput analysis of mitochondrial renewal and oxidative stress in cellular lifespan and quiescence of Schizosaccharomyces pombe

 描述(申请人提供)：干细胞是我们身体中最长寿的细胞之一，寿命可以超过几十年。它们对我们的长期健康至关重要，而与年龄相关的干细胞功能障碍与癌症、糖尿病和器官衰竭有关。这项建议的目标是确定干细胞和其他有丝分裂活性细胞如何维持细胞健康和避免过早衰老。干细胞的寿命包括增殖期和静止期(一种可逆的、可持续的不扩散细胞状态)。当增殖时，部分干细胞群必须取代分化或死亡的细胞。指导这一提议的首要假设是，细胞利用静止来减少线粒体在有氧呼吸过程中产生的活性氧物种(ROS)，以保持其复制寿命。这项提议的目的将阐明(1)静止是否可以通过减少细胞内ROS来延长细胞的寿命，以及(2)线粒体动力学(融合、裂变)是否 和分裂时的分布)在静止和增殖期间由细胞进行不同的管理，以实现完整的复制寿命。这项研究介绍了一种创新的高通量微流控装置，用于研究裂殖酵母的复制寿命，裂殖酵母是有丝分裂活跃的对称分裂细胞的经典模型。裂解酵母寿命显微分析器(Fylm)是一种微流控设备，它在数百个分裂酵母细胞的整个复制寿命内捕获和固定它们，同时将它们保存在一个恒定的、可控制的环境中，并以亚细胞分辨率进行观察。(1)利用ROS的Fylm和荧光ROS报告器，我将确定复制寿命与S.pombe静止后的增殖之间的相互作用。具体地说，我们将测试细胞在静止期间降低细胞内ROS水平以延长其复制寿命的假设。(2)接下来，我 将直接观察老化的线粒体在单个S.pombe细胞的复制寿命中的分布和动态。最后，线粒体融合和分裂将受到工程菌株的调控，以确定线粒体的总周转率或融合和分裂的比率是否对细胞在增殖和静止期间的健康至关重要。我假设，对称分裂的细胞在其整个复制生命周期内平均分割线粒体，而不是依靠快速的线粒体周转来移除功能失调的线粒体，在静止期间，融合和分裂的精确平衡维持线粒体的动态平衡。这些目标的完成将描绘出对称分裂和静止如何支持长寿、有丝分裂活跃细胞的真核模型中的长寿。最终，这些研究将阐明干细胞和其他长寿的真核细胞如何能够维持细胞健康。

 DESCRIPTION (provided by applicant): Stem cells are some of the longest-lived cells in our bodies, with a lifespan that can exceed dozens of years. They are critical to our long-term health, and age-related dysfunction in stem cells is linked to cancer, diabetes and organ failure. The objective of this proposal is to determine how stem cells and other mitotically-active cells maintain cell health and avoid premature aging. The lifespan of stem cells includes periods of proliferation and quiescence (a reversible, sustainable cell state of non-proliferation). When proliferating, part of the stem cell population must replace cells that differentiate or die. The overarching hypothesis guiding this proposal is that cells utilize quiescence to reduce reactive oxygen species (ROS) produced during aerobic respiration in mitochondria to preserve their replicative lifespan. The aims of this proposal will elucidate (1) if quiescence can extend the lifespan of cells by reducing intracellular ROS, and (2) if mitochondrial dynamics (fusion, fission and distribution at division) are managed differently by cells during quiescence and proliferation to achieve full replicative lifespan. This research introduces an innovative, high-throughput microfluidic device for studying replicative lifespan in the fission yeast Schizosaccharomyces pombe, a classic model for mitotically active, symmetrically dividing cells. The fission yeast lifespan microdissector (FYLM) is a microfluidic device that captures and immobilizes hundreds of fission yeast cells over their entire replicative lifespan, while they are kept in a constant, controllable environment and observed with subcellular resolution. (1) Using the FYLM and fluorescent ROS reporters of ROS, I will determine the interplay between replicative lifespan and proliferation after quiescence in S. pombe. Specifically, we will test the hypothesis that cell reduce intracellular ROS levels during quiescence to extend their replicative lifespan. (2) Next, I will directly observe the distribution and dynamics of aged mitochondria over the replicate lifespan of individual S. pombe cells. Finally, mitochondrial fusion and fission will be regulated n engineered strains to determine whether the overall turnover rate of mitochondria or the ratio of fusion to fission are critical to the cell health during proliferation and quiescence. I hypothesiz that symmetrically dividing cells equally divide mitochondria over their entire replicative lifespa, instead depending on rapid mitochondrial turnover to remove dysfunctional mitochondrial, and that during quiescence, precise balance of fusion and fission maintains mitochondrial homeostasis. Completion of these aims will delineate how symmetrical division and quiescence support longevity in a eukaryotic model for long- lived, mitotically active cells. Ultimately, thes studies will shed light on how stem cells and other long-lived eukaryotic cells are able to maintain cellular health.