The Role of Two Novel cGMP Binding Proteins in Dictyostelium

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Chemotaxis is the movement of cells up a chemical gradient. In Dictyostelium discoideum, chemotaxis is used under growth conditions to find bacteria as a food source, and under starvation conditions to promote aggregation of cells so that fruiting bodies may be formed. The basic mechanisms by which cells detect chemoattractants are conserved between Dictyostelium and mammalian cells, and the cellular machinery that drives cell migration is also conserved. Migration up a gradient involves the asymmetric remodeling of the cytoskeleton during the first minute after stimulation, and therefore requires that the activities and localization of key cytoskeletal components be tightly regulated in time and space. The long-term goal of this project is to understand the signaling pathways that control remodeling of the cytoskeleton during processes such as chemotaxis. The experimental tractability of Dictyostelium, coupled with its relative simplicity, make it an excellent model organism in which these fundamental signal transduction pathways can be worked out in detail. The general strategy is to focus on the observation that formation of an intracellular peak of cGMP is essential for chemotaxis in Dictyostelium cells. The hypothesis is that key cGMP-binding proteins in the cell directly or indirectly activate cytoskeletal components after treatment with chemoattractants. Despite the well-established importance of cGMP in signaling in chemotaxis, to date no cGMP-binding proteins have been purified or cloned in Dictyostelium. Two candidate cGMP-binding proteins have recently been identified by analyzing data from the Dictyostelium genomic database. Single and double gene disruption strains for these cGMP binding proteins will be engineered by homologous recombination, and the hypothesis that they play a central role in chemotaxis will be tested by filming cells as they respond to a micropipet filled with chemoattractant. The identification of cGMP binding proteins in Dictyostelium and determination of their role in chemotaxis will be a major advance in understanding how signaling during this process works. The project also provides the opportunity for undergraduate students to participate in independent research.