Functional Analysis of Ammonia Transporters during Development of Dictyostelium

盘状网柄菌是研究最简单的具有真正多细胞的真核生物之一。其天然食物来源的枯竭导致单细胞阿米巴的生长和细胞分裂的停止，启动了导致阿米巴合并为多细胞实体的发育程序。该计划的目标是产生和传播孢子，以便一些细胞在暂时不利的环境中生存下来。为了确保这一目标的实现，必须发生许多细胞分化事件和形态变化。有效的细胞与细胞之间和环境与细胞之间的通信和信号是成功实施发展方案所必需的。内源产生的氨被发育的网柄菌细胞用作自身产生的信号，不仅用于监测它们所处的环境，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。。该项目将探索一种理解氨信号的新方法，并将产生新的工具，使人们能够更集中地研究氨的功能以及实现这些功能的分子机制。竹鞭毛藻中有三种已知的氨转运蛋白(AMT)。将提出两个假设：1)不同细胞类型和发育结构微环境中氨水平的大部分差异是由于氨转运体的功能；2)AMTS作为氨传感器，将氨与信号转导途径联系起来，在氨信号中发挥直接作用。AMT基因的空间表达和编码的AMT蛋白在发育中的细胞和结构中的分布将被确定。将产生AMT基因被破坏的菌株，无论是单独的还是以各种组合的形式，这样就可以辨别每种AMT的具体作用。基于分布研究的结果和由此产生的突变菌株的表型，将建立每个AMT如何介导氨信号的可测试模型。将进行遗传、分子遗传和生化表征，以检验模型的预测，并确定调节AMTS功能的分子机制。由于氨在该生物体整个发育过程中的普遍使用，这项工作应该会导致对Dictyostelius多细胞发育程序的控制和调节有一个重要的、新的理解。虽然研究氨在其他系统中的传输是一个活跃的研究领域，但重点主要是从环境中吸收氨来满足细胞的氮需求。氨作为一种信号分子在发育过程中的更广泛作用几乎没有受到关注。在这方面，网柄金藻提供了一个很好的系统来探索内源产生氨的方式可以作为发育过程中的一种交流手段。拟议中的实验结果可能会影响人们对氨在其他生物体中扮演的角色的思考，并可能导致使用氨进行信号传递的新模型和影响，特别是在植物和哺乳动物的发育过程中。

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.