9307759 Fulton The ultimate goal of this laboratory is to elucidate the orchestration of causal pathways during cell differentiation. The paradigm employed for this purpose is the 100-minute change of Naegleria gruberi from amoebae to flagellates--from one eukaryotic motility form to another--a differentiation that is exceptionally accessible for analysis except for lack of molecular genetics. The quest continues via three ongoing studies, each of interest separately but all converging toward the goal. 1) Surprisingly this quick change has two "checkpoints," at 10 min and at 20 min, at which the strength of some signal(s) is evaluated and decisions are made whether to progress toward differentiation or to "hold." Experiments are planned to define these checkpoints. 2) Certain conserved proteins are synthesized during differentiation in processes that involve signals regulating gene expression, mRNA stability, and localization of these proteins in flagella and associated organelles. These proteins include four calcium-binding proteins: a flagellar and a cell-body calmodulin, calcineurin B and centrin (a.k.a. caltractin), which becomes associated primarily with the basal bodies when these organelles form de novo during differentiation. Studies are continuing to define these calcium- binding proteins by examining gene expression, sequence, and protein localization. 3) The essential tool of DNA-mediated transformation of Naegleria is being developed using an homologous selectable marker, the OMP decarboxylase gene. The description of checkpoints and the study of conserved localized proteins is expected to yield results of immediate interest while the molecular genetic system is achieved. Once gene introduction is possible in Naegleria, the information from the first two aims will make it feasible to determine causal pathways for signals from initiation of differentiation to assembly of components into basal bodies and flagellae. %%% This research takes advantage of a unicellular organism, Naegleria gruberi, to study the principles involved in cellular differentiation. In response to chemical, mechanical, or physical changes in its environment, this organism, within one hundred minutes, can change from an ameba to an actively swimming, streamlined form with two flagellae. These two forms move by two very different mechanisms which involve very different sets of cellular machinery . This quick change represents an excellent model to study the biochemical signal pathways and changes in gene expression involved in a well-defined developmental process. The results should shed light on the mechanisms of cellular differentiation common to all living things, and will also expand our knowledge of the diversity of mechanisms by which a variety of organisms respond to environmental signals. ***
