Functional Analysis of Ammonia Transporters during Development of Dictyostelium

Vanderbilt University, Nashville TN

Investigators

Abstract

0234254 Singleton Dictyostelium 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.