Our proposed research has four major goals, which we believe are attainable during the project period. All utilize the synchronous, hour-long differentiation of Naegleria from amebae to flagellates, which involves a dramatic change in cell phenotype, including the morphogenesis of new organelles. The first three goals, closely interrelated, are based on our observation that the tubulin that forms the flagellar microtubules is selectively synthesized de novo during differentiation and is immunochemically distinct from the tubulin that pre-exists in amebae. 1. Multi-tubulin hypothesis. Determine the nature of the differences between tubulins of Naegleria by continuing our immunochemical study, by selective analysis of the primary structure of tubulins from different structures, and by directly measuring the number of tubulin genes. 2. Regulations of flagellar tubulin synthesis. Determine whether flagellar tubulin mRNA is synthesized de novo during differentiation. Use purified mRNA to obtain cDNA and clone it in a plasmid vector. Use the cloned DNA to study the structure and expression of tubulin genes. 3. Spatial and temporal regulation of events from synthesis of flagellar tubulin to assembly into flagella. Determine whether there is an orderly relationship between synthesis and assembly. 4. Intracellular regulation of cell shape and motility. This goal is based on our discovery of psi, a cell-produced chemical factor that causes shape changes, and which is postulated to regulate intracellular release of calcium ions. Identify psi, study its mode of action and the "compartmentalization" of psi that occurs during differentiation.