The freeze-etch electron microscopic techniques developed in this laboratory will be used to study clathrin-mediated endocytosis in fibroblasts and macrophages. Questions to be addressed include: (1) what role do clathrin lattices play in bringing about receptor clustering? (2) what is the mechanism by which clathrin lattices curve into spheres and pinch off from the plasma membrane? and (3) how is clathrin recycled from internalized membranes back to the cell surface for additional round of endocytosis? Ongoing studies of all three processes are described to demonstrate the unique contributions that the quick-freeze, deep-etch, rotary=replication technique can make to this analysis. By permitting direct visualization of individual membrane receptors on the outsides of cells, as well as individual clathrin lattices on their insides, the technique has already demonstrated a number of correlations between clathrin lattice dynamics and changes in receptor distribution. By also permitting 3-D visualization of small clathrin polymers and individual clathrin """"""""triskelia"""""""" adsorbed to mica, the technique has also begun to shed light on the molecular interactions that underlie clathrin lattice dynamics. A broad range of experiments is proposed to further probe the rules by which clathrin molecules self-assemble and carry out their biological functions. Some of these experiments will involve electron microscopic analysis of the effects of chemical treatments that stimulate or inhibit endocytosis in cultured cells, particularly the inhibitory effects of cytoplasmic acidification and the stimulatory effects of cytoplasmic alkalinization. In addition, new observations on the effects of such pH changes on endosome and lysosome movement and fusion will be actively pursued. Finally, the molecular basis of clathrin lattice dynamics will be analyzed by observing the patterns of molecular interaction that clathrin displays with itself and with other proteins in vitro, under different conditions that mimic the above in vivo situations. These studies will hopefully increase our basic understanding of how cells regulate their uptake of proteins and their intracellular membrane traffic.
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