This project focuses on the molecular dynamics of an epithelial-mesenchymal transition (EMT). Primary mesenchyme cells (PMCs) in sea urchin embryo undergo an EMT at a highly predictable time easily observed in this transparent embryo. As they ingress micromere progeny become motile, change a number of adhesive properties and invade the blastocoel by passing through the basal lamina. Methods have been established for obtaining PMCs prior to, during, or just after ingression. At least five cell-cell or cell-substrate adhesive changes occur within 20-30 mm as the epithelial cell converts to a mesenchymal cell. At the transition the epithelial adhesion molecules are rapidly endocytosed and the mesenchymal adhesion molecules are exocytosed. Surprisingly, much, if not all the epithelial membrane is replaced during the transition. Since the transition of PMCs at ingression takes only about 20 min this suggests that the EMT involves a bulk turnover of plasma membrane. During the past funding period we cloned cadherin, alpha-catenin, beta-catenin, two integrins, moesin, Rho, Ras, and two substrate molecules, all of which are associated with the EMT. Assays have been developed for examining motility changes, adhesion changes, exocytosis, endocytosis, and altered cytoskeletal changes that occur during this crucial 20-30 min. These approaches will be used to address four specific aims: I. The dynamics of membrane turnover will be studied in detail to learn whether all plasma membrane molecules are internalized at EMT as suggested by the preliminary observations, or whether there is a rapid removal of epithelial adhesion molecules only. 2. The roles of beta-catenin, Rho, Ras and moesin will be studied to determine how these molecules assist in the five adhesion changes and the onset of mesenchymal cell motility, all of which occur within the 20-min EMT period. 3. Modified adhesion molecules will be expressed to determine the interdependence or autonomy of the five adhesion mechanisms that change at BMT. 4. A number of post-specification, pre-ingression changes have been observed in micromeres. In vitro and in vivo experiments will ask which of these are important for preparation of the EMT.
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