All eukaryotic cells communicate with their surroundings by taking up molecules from the plasma membrane. This uptake, known as endocytosis, can fulfill a variety of requirements including nutrition and recycling of the plasma membrane itself. A cell can use several different modes of endocytosis. Although many proteins involved in endocytosis have been discovered, knowledge of the precise mechanisms underlying endocytosis, and understanding the regulation and coordination of multiple pathways are still fragmentary. This project is aimed at dissecting two pathways of uptake in the ciliated protozoan Tetrahymena thermophila. The first pathway is specialized for nutrition, and allows these cells to take up large particles, such as bacteria, by a process that may resemble particle uptake in human immune cells. The second pathway has been less well described, but it may be involved in membrane recycling. Preliminary data suggest that both pathways appear to share some proteins, in particular a protein called dynamin. This was a surprising result because the function of dynamin in endocytosis was previously believed to be restricted to higher eukaryotic cells. A first aim of this project is therefore to confirm the localization of dynamin in Tetrahymena by using electron microscopy. To determine whether dynamin affects cellular morphology, electron microscopy will be performed on cells in which the dynamin gene has been disrupted. A second aim is to analyze the roles of dynamin and distinguish between direct and indirect effects following loss of dynamin function. This aim will be achieved by replacing dynamin with mutagenized variants that are sensitive to rapid temperature-triggered inactivation. A third aim is to understand how dynamin is accurately targeted to its site of action. This will be investigated by creating mini-proteins consisting of small parts of the dynamin protein, and asking which of these contains sufficient information for proper localization. This project will provide research training for graduate and undergraduate students.
