The quick-freeze, deep-etch technique developed in this laboratory will be used to study three key processes in eukaryotes, namely, the formation and dissolution of clathrin cages, the assembly and disassembly of an extracellular matrix, and recognition between gametic cells. Ongoing studies of all three systems are described to demonstrate the unique contributions the quick-freeze, deep-etch technique can make to their understanding. Critical is the ability of the technique to visualize proteins in their native state and after experimental manipulation. Thus, for example, the components of the clathrin cages can be viewed as individual proteins (""""""""triskelions"""""""", """"""""cagin"""""""", etc.) or in various stages of assembly, disassembly, and association with membrane surfaces. Particularly valuable are images of interactions between fibrous proteins, such as those that constitute the extracellular matrix and the sexual agglutinins of Chlamydomonas, since these are impossible to decipher by any other electron microscopic technique. A broad range of experiments is proposed to probe the """"""""rules"""""""" by which these self-assembling sets of proteins carry out their biological functions. Many of these experiments will involve the structural analysis of proteins purified in two collaborating laboratories; in addition, new approaches are described for studying such processes as clathrin/membrane interactions. These studies will hopefully increase our basic understanding of how extracellular proteins interact, how cells regulate their uptake of proteins and their internal membrane traffic, and how cells recognize each other.
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