Microfluidic Devices for Studying the Development and Aging of Bacteria

 描述(申请人提供)：最近的研究表明，衰老不仅在真核生物中起作用，在细菌中也是如此。这一发现挑战了数十年来的进化理论，该理论将原核生物区分为不受衰老影响的永生细胞谱系。更流行的理论认为，细菌老化是为了减轻累积损伤的后果而进化的。通过分裂时损伤的不对称分离，老化的细胞通过遗传损伤经历有害的影响，从而使其相对无损伤的对应物恢复活力。因此，该模型认为，对损害控制的选择推动了不对称繁殖和衰老的同时进化，衰老应该作为一种共同的生活史策略来进化。最近的观察发现，即使在形态对称分裂的细菌中也存在衰老，这表明潜在的生殖不对称确实可能发生在基本上所有的细胞生物体中。我们假设，细菌的不对称繁殖是通过逐渐积累老化细胞中的损伤来驱动衰老的。我们将在不对称细菌、新月弯杆菌和各种不对称突变体的主要模型中检查衰老。荧光显微镜将监测蛋白质聚集和氧化损伤以及细胞活力的测量，从而探索细菌老化效应的近端机制。使用抗生素和氧化应激源的不同损伤率将评估特定突变体隔离和耐受累积损害的能力。我们预测，破坏正常的生殖不对称将减缓衰老过程，同时降低整个人群的适合度，并增加对损伤的敏感性。用现有的方法来测试损伤、老化和不对称性之间的假设联系尤其困难。衰老表现为生命活力的衰退。 老化细胞的历史参数(例如，细胞延长率、分裂率和存活率)与其恢复活力的对应物进行比较。因此，衰老研究需要对多代细胞及其谱系进行详细测量，以便获得足够的统计能力。因此，我们正在开发具有集成纳米通道阵列的微流体设备，以便在更长的时间段内观察老化的细菌谱系。纳米通道限制细菌在单一维度上的生长，纳米通道两端的微流体通道引导新鲜介质的持续输入，同时冲洗掉生长在纳米通道之外的细胞。这样的设计可以直接、高分辨率地观察最年轻的细菌(在纳米通道中心丰富)及其几代人的直系后代--这是衰老研究的关键细胞。虽然我们最初的研究重点是硫杆菌，但这种微流控设备的设计可以作为一个通用平台来研究不同细菌中的衰老和其他表观遗传现象。该项目的具体目标是测量新月藻的衰老及其影响因素，探索不对称性与衰老之间的联系，并确定细菌损伤控制策略的可塑性。

 DESCRIPTION (provided by applicant): Recent research reveals that aging acts not only in eukaryotes, but also in bacteria. This revelation challenges decades of evolutionary theory, which held prokaryotes apart as immortal cell lineages immune to aging effects. More current theory suggests that bacterial aging evolved in order to mitigate the consequences of accumulated damage. Through asymmetric segregation of damage upon division, an aging cell experiences deleterious effects via inherited damage, thereby rejuvenating its relatively damage-free counterpart. This model thus argues that selection for damage control drives the concurrent evolution of asymmetric reproduction and aging, and that aging should evolve as a common life history strategy. Recent observations find aging even in bacteria with morphologically symmetric division, indicating that underlying reproductive asymmetry may indeed occur in essentially all cellular organisms. We hypothesize that asymmetric reproduction in bacteria specifically drives aging via progressive accumulation of damage in aging cells. We will examine aging in the predominant model for asymmetric bacteria, Caulobacter crescentus and various asymmetry mutants. Epifluorescence microscopy will monitor protein aggregation and oxidative damage alongside measures of cell vitality, thereby exploring the proximal mechanism of bacterial aging effects. Varying damage rates using antibiotics and an oxidative stressor will evaluate the ability of specific mutants to segregate and tolerate accumulated damage. We predict that disrupting normal reproductive asymmetry will attenuate the aging process, while at the same time reducing population-wide fitness and increasing susceptibility to damage. Testing the hypothesized link between damage, aging, and asymmetry proves particularly difficult with existing methodology. Aging manifests itself as a decline in vital life history parameters (e.g., cell elongation rate, division rate, and survival) of old cells compared o their rejuvenated counterparts. Aging studies thus require detailed measurements of many cells and their lineages over multiple generations in order to achieve adequate statistical power. We are therefore developing microfluidic devices with integrated nanochannel arrays that permit observation of aging bacterial lineages over extended time periods. The nanochannels constrain growth of the bacteria along a single dimension, and microfluidic channels on each end of the nanochannels direct constant input of fresh media while washing away cells that grow beyond the nanochannels. Such a design permits direct, high-resolution observation of the youngest bacteria (enriched at the nanochannel center) and their immediate descendants over several generations - the key cells for aging studies. Although focused on Caulobacter for our initial studies, this microfluidic device design serves as a general platform to study aging and other epigenetic phenomena in diverse bacteria. The specific aims for the project are to measure aging and its effectors in C. crescentus, explore the connection between asymmetry and aging, and determine the plasticity of bacterial damage control strategies.