Minority Predoctoral Fellowship Program

描述（由申请人提供）：肠道Na+/H+交换器NHX-2调节线虫C的细胞pH值和营养摄取。功能丧失导致脂肪储存减少和寿命延长。为了询问这些表型是否是改变的pH或热量限制的机械结果，进行了改变肠细胞pH的其他靶标的全局RNAi筛选。几乎四分之一的45个克隆确定功能的线粒体;特别是，ETC复合物I是很好的代表，因为是蛋白质参与线粒体！核糖体功能和调节线粒体形态。进一步的测试表明，几个克隆至少部分地表现出细胞非自主性。我们在这里首先提出使用细胞特异性RNAi的新系统来鉴定哪些细胞中发生非自主功能。然后，我们将检查相关细胞中RNAi后的线粒体氧化还原电位，pH值，形态和体内能量产生，以及目标蠕虫的脂肪摄取和寿命。最后，我们将在培养的线虫胚胎细胞中靶向每个克隆，以直接测试细胞自主性。这些实验的结果将为线粒体如何影响肠道细胞pH值以及这种耦合如何影响脂肪代谢和衰老提供机制见解。 在过去十年中，肥胖已成为一个紧迫的健康问题，特别是在年轻人中，并且是发展糖尿病和心脏病的主要风险因素之一。我们的研究旨在确定满足细胞能量需求的线粒体如何影响保守遗传模式生物中的pH稳态。由于细胞的pH值与营养吸收、脂肪积累和寿命有关，这种功能性偶联可能代表了细胞通讯的独特方式和抗肥胖治疗的新靶点。

DESCRIPTION (provided by applicant): The intestinal Na+/H+ exchanger NHX-2 regulates both cellular pH and nutrient uptake in the nematode C. elegans, and loss-of-function leads to decreased fat stores and increased lifespan. In order to ask whether these phenotypes are mechanistically a result of altered pH or of caloric restriction, a global RNAi screen for other targets that alter intestinal cellular pH was performed. Almost a quarter of the 45 clones identified function in the mitochondria; in particular, ETC complex I was well-represented, as were proteins involved in mitochondria! ribosome function and in regulating mitochondrial morphology. Further testing suggested that several clones act at least in part cell non-autonomously. We propose here first to identify in which cells the non-autonomous functions occur using a novel system for cell specific RNAi. Then we will examine mitochondrial redox potential, pH, morphology, and energy production in vivo following RNAi in the relevant cells, as well as fat uptake and longevity in the targeted worms. Finally, we will target each clone in cultured nematode embryonic cells to directly test cell autonomy. The results of these experiments will provide mechanistic insights into how mitochondria influence intestinal cellular pH, as well as how this coupling influences fat metabolism and aging. Obesity has become a pressing health concern, particularly in younger people, over the last decade, and is one of the leading risk factors for developing diabetes and heart disease. Our research is aimed at defining how mitochondria, which meets the energy demands of the cell, influences pH homestasis in a well- conserved genetic model organism. Since cellular pH is linked to nutrient uptake, fat accumulation, and longevity, this functional coupling may represent a unique means of cellular communication and a novel target for anti-obesity therapeutics.