The goal of this project is to understand the endocytic mechanisms for routing ligands and receptors to different destinations and to determine how this routing is a determinant of physiological consequences. Receptor-mediated endocytosis is important in many biological processes, including uptake of LDL and delivery to lysosomes, internalization of transferrin and release of iron in acidified endosomes, clearance of signalling ligands such as insulin or EGF from the surface of cells, antigen presentation, infection by many types of viruses, and entry of certain toxins into cells. There is evidence that endocytosis and recycling is the mechanism for insulin-regulated redistribution of hexose transporters, -and it has been suggested that the defect in cystic fibrosis may alter endocytic trafficking. Mechanisms for targeting ligands and receptors after endocytosis are not clear. Even the most basic relationships among the organelles involved are not understood and agreed upon by researchers in the field. In this project, optical microscopy will be used to directly observe and measure endocytic processes. There have been dramatic increases in the power of microscopy for studying living cells which provide a unique opportunity to observe the trafficking of nearly native molecules in minimally perturbed intact cells. Bright, photostable fluorescent derivatives of natural ligands such as LDL and transferrin will be used to directly observe dynamic interactions among endocytic organelles using confocal microscopy and image intensification fluorescence microscopy. Digital image analysis will be used to quantify the kinetics of ligands moving into and out of individual,identified endocytic organelles. Properties of the organelles such as their lifetimes and rate of fusion with other endosomes will be quantified. In addition to studying natural ligands and receptors, the behavior of fluorescent lipids, GPI-anchored proteins, modified receptor proteins, and multivalent ligand-receptor complexes will be studied to understand the molecular determinants for trafficking of membrane components. The role in endocytic trafficking of specific proteins such as cytoskeleton-associated motors or small GTP-binding proteins will be tested in living cells by microinjection of antibodies, transfection with normal and altered proteins, and analysis of mutant cell lines. It is expected that these studies will lead to a fairly complete map of the endocytic pathway in nonpolarized cells within five years. These studies should help specialists in the field of endocytosis by providing an improved basis for understanding and interpreting in vitro experiments. They should also aid nonspecialists who are interested in determining how endocytosis might be involved in a variety of biological processes.
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