Functional Analysis of Ammonia Transporters during Development of Dictyostelium

盘基网柄菌发育过程中氨转运蛋白的功能分析

• 批准号: 0234254
• 负责人: Charles Singleton
• 金额: $ 50万
• 依托单位: Vanderbilt University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0234254&HistoricalAwards=false
• 关键词: Functional; Analysis; Ammonia; Transporters; during

0234254SingletonDictyostelium discoideum is one of the simplest studied eukaryotes that possesses true multicellularity. Depletion of its natural food source results in a cessation of both growth and cell division of the unicellular amoebae, initiating a developmental program that leads to the coalescence of the amoebae into a multicellular entity. The goal of the program is to produce and disperse spores in order that some cells survive the temporary unfavorable environment. A number of cell differentiation events and morphological changes must occur to ensure the attainment of this goal. Efficient cell-to-cell and environment-to-cell communication and signaling are necessary for the successful implementation of the developmental program. Endogenously produced ammonia is used by developing Dictyostelium cells as a self-generated signal for monitoring not only their environment, but also the morphological changes and the differentiation events they are undergoing, and thus their progression through the developmental program. . This project will explore a novel approach to understanding ammonia signaling and will generate new tools that will allow a more focused study of the functions of ammonia and the molecular mechanisms used to carry out those functions. There are three known ammonia transporters (AMTs) in Dictyostelium. Two hypotheses will be addressed: 1) much of the differences in ammonia levels within various cell types and within the microenvironments of the developing structures is due to the function of ammonium transporters; and 2) the AMTs play a direct role in ammonia signaling by serving as ammonia sensors that link ammonia to signal transduction pathways. The spatial expression of the amt genes and the distribution of the encoded AMT proteins within the developing cells and structures will be determined. Strains will be generated in which the amt genes are disrupted, either singly or in various combinations, so that the specific role of each AMT can be discerned. Based on the results of the distribution studies and the resulting phenotypes of the mutant strains, testable models of how ammonia signaling is mediated by each AMT will be formulated. Genetic, molecular genetic, and biochemical characterizations will be carried out to test the predictions of the models and to determine the molecular mechanisms that mediate the function of the AMTs. Because of the pervasive use of ammonia throughout development of this organism, the work should lead to a significant, new understanding of the control and regulation of the multicellular developmental program of Dictyostelium. Although studying ammonia transport in other systems is an active area of research, the focus is mostly on uptake of ammonia from the environment to supply the cell's nitrogen needs. A broader role for ammonia as a signaling molecule used during development has received little attention. In this regard, Dictyostelium provides a good system for exploring the ways endogenously produced ammonia can serve as a means of communication during development. The findings from the proposed experiments may impact thinking on the role ammonia plays in other organisms and may lead to new models of, and implications for, the use of ammonia in signaling, particularly during plant and mammalian development.

0234254 SingletonDictyosteelium discoideum是研究最简单的真核生物之一，具有真正的多细胞性。其天然食物来源的耗尽导致单细胞阿米巴的生长和细胞分裂停止，启动导致阿米巴合并成多细胞实体的发育程序。 该计划的目标是产生和分散孢子，以便一些细胞在暂时的不利环境中存活下来。 必须发生许多细胞分化事件和形态变化才能确保实现这一目标。 有效的细胞与细胞和环境与细胞的通信和信号传导是成功实施发展计划所必需的。内源性产生的氨被发育中的网骨藻细胞用作自生信号，不仅用于监测它们的环境，还用于监测它们正在经历的形态变化和分化事件，从而监测它们在发育过程中的进展。 .该项目将探索一种新的方法来理解氨信号，并将产生新的工具，使氨的功能和用于执行这些功能的分子机制的研究更加集中。 在网骨藻中有三种已知的氨转运蛋白（AMTs）。 将解决两个假设：1）在各种细胞类型内和发育结构的微环境内氨水平的大部分差异是由于铵转运蛋白的功能;和2）AMTs通过充当将氨连接到信号转导途径的氨传感器而在氨信号传导中发挥直接作用。 将确定amt基因的空间表达和编码的AMT蛋白在发育细胞和结构内的分布。 将产生其中amt基因单独或以各种组合被破坏的菌株，使得可以辨别每种AMT的特定作用。 基于分布研究的结果和突变菌株的表型，将制定氨信号传导如何由每个AMT介导的可测试模型。 将进行遗传、分子遗传和生化表征，以测试模型的预测并确定介导AMC功能的分子机制。 由于氨在这种生物体的整个发育过程中的普遍使用，这项工作应该会导致对Dictyosteoblastoma的多细胞发育程序的控制和调节的重要的新的理解。 虽然研究氨在其他系统中的运输是一个活跃的研究领域，但重点主要是从环境中吸收氨以供应细胞的氮需求。 氨作为发育过程中使用的信号分子的更广泛的作用很少受到关注。 在这方面，Dictyosteroids提供了一个很好的系统，探索内源性产生的氨可以作为一种手段，在发展过程中的通信。 从拟议的实验结果可能会影响对氨在其他生物体中所起作用的思考，并可能导致新的模型，并影响，氨在信号中的使用，特别是在植物和哺乳动物的发展。