The overall goal of this project is to understand how ligands, receptors, membrane lipids and solute molecules move through cells following endocytosis. This involves studies of the relationships among endocytic compartments and studies of the molecular mechanisms that govern traffic. In order to carry out these studies optical microscopy, electron microscopy, and a variety of biophysical and biochemical methods are used. The first goal is to obtain a quantitative description of the kinetics of sorting in endocytic pathways. Using quantitative fluorescence microscopy with several different markers, trafficking kinetics and efficiencies of sorting among organelles including sorting endosomes, the endocytic recycling compartment, the TGN, late endosomes and the plasma membrane will be measured. The effects of expressing modified proteins that affect trafficking will be examined to determine the precise site of their action. The effects of changes in membrane lipid composition and the role of transmembrane domains in trafficking of membrane proteins will also be examined. These studies will extend significantly previous kinetic studies of the endosomal system. A second goal is to analyze the structure organization and function of the endocytic recycling compartment. This will include a high resolution 3-D electron microscopic analysis of the structure of this compartment as well as studies of the proteins that regulate its organization and dynamics. The third goal is to analyze the membrane organization and trafficking of sterols and other lipids. This will include observing the distribution of lipids on the plasma membrane under various conditions and correlating changes in distribution with changes in endocytic trafficking of lipids. These studies will lead to an increasingly complete description of endocytic trafficking pathways.
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