Microfluidic Devices for Studying the Development and Aging of Bacteria

 描述（由申请人提供）：最近的研究表明，衰老不仅在真核生物中起作用，而且在细菌中也起作用。这一发现挑战了几十年来的进化理论，该理论认为原核生物是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，衰老细胞通过遗传损伤经历有害作用，从而使其相对无损伤的对应物恢复活力。因此，这个模型认为，损害控制的选择驱动不对称生殖和老化的同时进化，老化应该演变为一个共同的生活史策略。最近的观察发现，即使在形态对称分裂的细菌中也会发生衰老，这表明基本上所有细胞生物都可能发生潜在的生殖不对称。我们假设细菌的不对称繁殖通过衰老细胞中损伤的渐进积累来特异性地驱动衰老。我们将在不对称细菌、新月柄杆菌和各种不对称突变体的主要模型中研究衰老。落射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，从而探索细菌衰老效应的近端机制。使用抗生素和氧化应激物改变损伤率将评估特定突变体分离和耐受累积损伤的能力。我们预测，破坏正常的生殖不对称将减弱衰老过程，同时降低整个种群的适应性，增加对损伤的敏感性。用现有的方法来测试损伤、老化和不对称之间的假设联系是特别困难的。衰老表现为生命力的衰退 历史参数（例如，细胞伸长率、分裂率和存活率）。因此，衰老研究需要详细测量多代细胞及其谱系，以获得足够的统计功效。因此，我们正在开发具有集成纳米通道阵列的微流体装置，其允许在延长的时间段内观察老化的细菌谱系。纳米通道限制细菌沿着单一维度生长，并且纳米通道的每一端上的微流体通道引导新鲜培养基的恒定输入，同时洗掉生长超过纳米通道的细胞。这种设计允许直接，高分辨率观察最年轻的细菌（在纳米通道中心富集）及其几代的直系后代-衰老研究的关键细胞。虽然我们最初的研究集中在柄杆菌，但这种微流体装置设计可作为研究不同细菌中衰老和其他表观遗传现象的通用平台。该项目的具体目标是测量C. crescentus，探索不对称和衰老之间的联系，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.